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Nanomaterials Based Electrochemical Approaches for
Biosensing and Bacterial Disinfection
Bal Ram Adhikari
PhD candidate Biotechnology
o
Advisor: Dr. Aicheng ChenAdvisor: Dr. Aicheng Chen
o
Co-advisor: Dr. Heidi Schraft
Co-advisor: Dr. Heidi Schraft
o
Committee member: Dr. Neelam KhaperCommittee member: Dr. Neelam Khaper
o
External examiner: Dr. Antonella Badia
External examiner: Dr. Antonella Badia
o
Committee chair:Committee chair: Dr. Wely FlorianoDr. Wely Floriano
PhD Dissertation defense
1. Introduction
2. Background and Rational
3. Research objectives
4. Experimental details
5. Results and Discussions
6. Summary and Future prospectus
7. Acknowledgements
Outline of presentation
Applications
Electrochemical
approaches
Detection of
pathogens ( bacteria,
viruses)
Testing of bloods
( biochemistry)
Quality control
monitoring
Contamination detection
Food and drug process
monitoring
Diagnosis of plant and
animal diseases
Monitoring of chemicals
Quality control of meat
and plant products
Environmental
Detection of toxic
chemicals in air, water
and soil
Pollutants degradation
and bacterial disinfection
R.S. Sethi, Biosens. Bioelectron. 9, 243 (1994).
Clinical/Medical
Industrial
Agriculture
Role of nanomaterials in electrochemistry
Any particle size in between 1-100 nm are
nanomaterials
Increase the surface/volume ratio
Reduced distances e.g. between immobilized
biomolecules and electrodes- lower the response
time
Enhances the production of Reactive oxygen
species (ROSs) sufficient to disinfect bacteria and
organic compounds
Similar size with biomolecules cause -intracellular
tagging and ideal for bioconjugation
Y. Wang, Z. Tang, N.A. Kotov. Materials Today. 8, Issue 5, Supplement 1, 20 (2005).
W. Kulisch, R. Freudenstein (Eds.), p. 3, Springer Verlag, Dordrecht, The Netherlands (2009)
Contd..
Graphene is a two-dimensional (2D), single-
layer sheet of Sp
2
-hybridized carbon atoms that
are closely packed into a hexagonal lattice
structure. (small)
Chemical reduction methods vs green methods
for preparation of graphene
Study on nanocomposite behaviour of
graphene materials with single walled carbon
nanotubes- new level of catalytic response
Contd..
Further exploring the application of nanostrucutred materials; the
properties of TiO
2
have been investigated extensively for
photoelectrochemical bacterial disinfection. (xin 19-20)
It is promising photocatalyst due to low cost, high photocatalytic
activity, and chemical stability (17- chen paper)
A variety of electrocatalysts for anode materials including carbon, Pt,
PbO
2
, IrO
2
, SnO
2
, Pt-Ir, and boron-doped diamond electrodes have
been extensively investigated for electrocatalytic oxidation (Chen
paper)
Rationale of thesis
Global Analgesics Market of AP US$34.6 billion and valacyclovir $4.
billion by 2017
Pharmacopeia study during drug formulations are time consuming and
expensive.
The estimated incidence of annual hospitalization for acetaminophen
overdose in Canada is 27 to 46 per 100,000 persons.
The increasing R & D investment and incidence of acetaminophen induced
hepatotoxicity demand the urgent need of reliable and easy to operate sensor
One step electrochemical reduction and the deposition of graphene oxide
(GO) on an electrode surface- a very quick and unique sensor fabrication
technique with very small amount of GO.
Patients screening
Pharmaceutical
formulations
Bioavailability
testing
Rationale contd..
The partial reduction of graphene oxide (ERG) is advantageous for enhanced
electrocatalytic activity and the attachment of biomolecules through π-π interactions,
in contrast to CRG
Entrapment is one of the primary approaches for enzyme immobilization; however, it
suffers from a few critical drawbacks, including leakage and high mass transfer
resistance to substrates. SWCNTs–rGO nanohybrid thin film has been utilized as
platform for the polymer based enzyme immobilization- great biocompatibility with
high activity.
New level of catalytic activity achieved through the combining approach of
nanomaterials e.g. SWCNTs–rGO nanohybrid for biosensing; photocatalyst
(nanoporous TiO 2
) and electrocatalyst (RuO 2
) for bacterial disinfection.
Bifunctional approach of water disinfection: a very quick and efficient bacterial
disinfection in comparison to existing methods.
Experimental set up
CHI 660D for electrochemical
workstation
Three electrode system for analytical
measurements
Electrochemical Methods
Cyclic Voltammetry
Differential pulse voltammetry Chronoamperometry
A. Chen, B. Shah, Anal. Methods 5 (2013) 2158-
Tools used for characterization
Scanning electron microscopy (SEM)
Energy dispersive X-ray spectroscopy (EDS)
X-ray diffraction (XRD)
RAMAN spectroscopy
Fourier transform infrared spectroscopy (FTIR)
Confocal laser microscopy for live dead bacterial analysis
Non-pyrogenic sterilized 96 well cell culture microtiter plates
LIVE/DEAD® Bac Light™ bacterial viability kit
Qproteome
TM
Bacterial Protein Preparation Kit
Nanodrop instrument
1
H NMR
TOC analyzer
OH
COOH
COOH COOH
O
O
OH
OH COOH
OH
E vs ( Ag AgCl) / V
-1.5 -1.0 -0.5 0.0 0.
I /
**-
-**
0
1st cycle
3rd cycle
5th cycle
Methodology: Sensor design
Graphene oxide
(Commercial)
Electrochemical reduction process ( 10mV/s)
in PBS (pH 7.4)- 0.3mg/mL GO
Reduced Graphene oxide
EDX spectra
SEM image of
deposited rGO
Tablet used from Thunder
bay regional hospital
Cyclic voltammetric measurements:AP
E / V (Ag/Agcl)
0.0 0.1 0.2 0.3 0.4 0.5 0.
I /
**-
-**
0
2
4
6
8
10
12
a.
b.
c.
At 20 mV/s in 250 μM AP + 0.1 M 20 mL PBS (pH
a.Bare GCE
b.ERG/GCE
c.ERG/GCE without AP
N-acetyl-p-aminophenol (AP)
oxidized to N-acetyl-p-
benzoquinone imine (NAPQI)-
reversible process
Analytical Detection:AP
Successive addition (5-800 μM) AP in 0.1 M
PBS
E/V(Ag/AgCl)
0.2 0.3 0.4 0.5 0.
I/
0
2
4
6
8
10
12
14
16
18
5
50
100
800
a.
[ Acetaminophen ] / μM
0 200 400 600 800
I / μA
0
2
4
6
8
10
12
14
R
2 =0.
b.
Time / Sec
0 200 400 600 800
I /
A
0.
0.
0.
0.
0.
0.
0.
5nM
0.2 M
2
a.
[Acetaminophen] / nM
0 1000 2000 3000 4000 5000
I /
0.
0.
0.
0.
0.
0.
0.
R
2 = 0.
b.
Succesive addition of 5nm, 0.2 μM and 2μM
AP in 0.1 M PBS; E app
:0.5V
LOD : 2.013 nM
(A) DPVs recorded in 0.1 M PBS (pH 7.4) + 20mM acetaminophen without interferents (a) and
in the presence of 40mM each ascorbic acid (b), uric acid (c), adenine (d), glucose (e), sucrose
(f) and the mixture of all these biomolecules (g). (B) Relative anodic peak current
Interference and real sample analysis of developed sensor on AP detection
Concentration spiked/μM Concentration detected/μM % Recovery
10.00 10.32 103.
20.00 19.80 98.
25.00 24.02 96.
Recovery tests of generic 325 mg acetaminophen tablets in human serum plasma.
