biotechnology of aminophenol PhD defenseppt.ppt

Nanomaterials Based Electrochemical Approaches for
Biosensing and Bacterial Disinfection
Bal Ram Adhikari
PhD candidate Biotechnology
1
o Advisor: Dr. Aicheng Chen
o Co-advisor: Dr. Heidi Schraft
o Committee member: Dr. Neelam Khaper
o External examiner: Dr. Antonella Badia
o Committee chair: Dr. 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
2
Outline of presentation

Introduction
3
 Electrochemical approaches are considered a
physicochemical discipline with wide-
ranging applications that are useful in our
daily life e.g. sensing to disinfection (Ota et
al. 2014)
 Due to their point-of-care applications,
electrochemical approaches are the most
promising candidate technologies.
 Sensors are devices which capture the
physical, chemical and biological changes
and convert changes into measurable signal
 Electrochemical biosensor contain a
biological recognition element (enzymes,
proteins, antibodies, nucleic acids) reacts
with target analytes and produce an electrical
signal.
substrate product
Reporter
(enzyme)
Apply voltage
Measure current prop.
to concentration of substrate
Electrode ( Transducer)
N. Ronkainen, H. Halsall, W Heineman. Electrochemcial biosensors. Chem. Soc. Rev., 2010, 39, 1747-1763

Applications
4
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
5
 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)

6
Background and Rational of thesis
 Carbon materials, an element in nature, has been
recognised by humans for a long time.
 Diverse allotropes of CNs from zero-dimensional
(0D) to three-dimensional (3D).1,2-nanoscale
 Carbon nanomaterials; graphene, carbon
nanotubes (CNTs), carbon dots (CDs), carbon
nanofibers (CNFs), nanodiamonds (NDs) and
fullerenes (C60) have been extensively used as
electrode materials for sensor design (nanoscale)
 CNs have intrinsic electrochemical activity, high
electrical conductivity, large surface area, ease of
functionalisation and biocompatibility.
(Nanoscale)
 Low cost of fabrication, high stability, fast
response time and specific detection of analytes
are the key requirement of the biosensor design.

7
Contd..
 Graphene is a two-dimensional (2D), single-
layer sheet of Sp2-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

8
Contd..
 Further exploring the application of nanostrucutred materials; the
properties of TiO2 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,
PbO2, IrO2, SnO2, Pt-Ir, and boron-doped diamond electrodes have
been extensively investigated for electrocatalytic oxidation (Chen
paper)

9
 Quantitative analysis of pharmaceuticals is essential during drug
development and clinical trial phase for monitoring bio-availability,
pharmacokinetics and possible drug abuse
 Acetaminophen (AP) and valacyclovir are the extensively used
analgesic and antiviral drugs.
 Global Analgesics Market of AP US$34.6 billion and valacyclovir
$4.8 billion by 2017.
 The estimated incidence of annual hospitalization for
acetaminophen overdose in Canada is 27 to 46 per 100,000 persons
Contd..

10
Rationale of thesis
 Global Analgesics Market of AP US$34.6 billion and valacyclovir $4.8
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

11
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 TiO2) and electrocatalyst (RuO2) for bacterial disinfection.
 Bifunctional approach of water disinfection: a very quick and efficient bacterial
disinfection in comparison to existing methods.

12
Research objectives
Objective 1: Study the synthesis, characterization and optimization of
carbon based nanomaterials for electrochemical sensing/biosensing
Objective 2: Study the preparation and analytical performance of
reduced graphene oxide (rGO) towards detection of acetaminophen
Objective 3: Optimize graphene oxide concentration and deposition cycle
for sensitive and simultaneous detection of valacyclovir and
acetaminophen.
Objective 4: Study the biocompatibility behaviour of rGO nanocomposite
in combination with single walled carbon nanotubes (SWCNTs)-alcohol
dehydrogenase (ADH) as model enzyme.
Objective 5: Investigate the synergistic effects of a photocatalyst
(nanoporous TiO2) and electrocatalyst (RuO2) to construct a bifunctional
electrode for a bacterial disinfection strategy.

Experimental set up
 CHI 660D for electrochemical
workstation
 Three electrode system for analytical
measurements
13
Electrochemical Methods
Cyclic Voltammetry Differential pulse voltammetry Chronoamperometry
 A. Chen, B. Shah, Anal. Methods 5 (2013) 2158-2173

14
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® BacLight™ bacterial viability kit
QproteomeTM Bacterial Protein Preparation Kit
Nanodrop instrument
1H NMR
TOC analyzer

15
Project 1: Sensitive Detection of Acetaminophen with
Graphene-Based Electrochemical Sensor

OH
COOH
COOH
COOH
O
O
OH
OH COOH
OH
E vs ( Ag AgCl) / V
-1.5 -1.0 -0.5 0.0 0.5
I
/

-60
-40
-20
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 16
Tablet used from Thunder
bay regional hospital

Cyclic voltammetric measurements:AP
17
E / V (Ag/Agcl)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
I
/

-6
-4
-2
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 7.4)
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

18
Optimization of sensor
(A) CVs - in 0.1 M PBS (pH 7.4) - 250mM AP from 20 to
125 mV/s scan rate
(B) Plots of the anodic and cathodic peak currents versus the
square root of the scan rates ( diffusion-controlled process)
DPVs - two-cycle (a), five-cycle (b) and ten-cycle (c)
electrodeposition of graphene measured in 0.1 M
PBS (pH 7.4) containing 250mM acetaminophen.

Analytical Detection:AP
19
Successive addition (5-800 µM) AP in 0.1 M
PBS
E/V(Ag/AgCl)
0.2 0.3 0.4 0.5 0.6
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
R2=0.9963
b.
Time / Sec
0 200 400 600 800
I
/
A
0.0
0.1
0.2
0.3
0.4
0.5
0.6
5nM
0.2M
2
a.
[Acetaminophen] / nM
0 1000 2000 3000 4000 5000
I
/

0.0
0.1
0.2
0.3
0.4
0.5
0.6
R2= 0.985
b.
 Succesive addition of 5nm, 0.2 µM and 2µM
AP in 0.1 M PBS; Eapp:0.5V
 LOD : 2.013 nM

20
(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 10.32 103.2
20.00 19.80 98.89
25.00 24.02 96.08
Recovery tests of generic 325 mg acetaminophen tablets in human serum plasma.

21
Conclusion
 Graphene based sensor has
been developed for
acetaminophen detection
 Very low detection limit (2.13
nM) and wide linear range of
detection (5 nM to 800 µM) has
been achieved
 Very high recovery rate in
human plasma sample with
potential of practical application
 Useful in the detection of
acetaminophen induced
hepatotoxicity B.-R. Adhikari, M. Govindhan, A. Chen. Electrochim. Acta,
2015, 162:198-204

22
Project 2: Simultaneous and Sensitive Detection of Acetaminophen and
Valacyclovir Based on Two Dimensional Graphene Nanosheets
Valacyclovir oxidation: two electron transfer process through intermediate (8-
oxovalacyclovir)-non reversible oxidation

23
Electrode fabrication: Methodology
0.3 mg/mL in PBS (pH-9)
Raman shift ( cm-1)
800 1000 1200 1400 1600 1800
Intensity
D
G
rGO
GO
E vs ( Ag AgCl) / V
-1.5 -1.0 -0.5 0.0 0.5
I
/

-60
-40
-20
0
1st cycle
3rd cycle
5th cycle
Valacyclovir obtained
from Thunder bay regional
hospital

24
Optimization of sensor for valacyclovir detection
Electrodeposition cycle
2 4 6 8 10 12 14 16
J
/
cm
-2
1
2
3
4
5
6
7
GO / mg mL-1
0.0 0.2 0.4 0.6 0.8 1.0 1.2
J
/
A
cm
-2
0
20
40
60
80
100
120
Peak
potential
range
1.00
1.02
1.04
1.06
1.08
1.10
Peak current
Peak potential
B
E vs ( Ag / AgCl) / V
0.7 0.8 0.9 1.0 1.1 1.2 1.3
J
/
cm
-2
0
20
40
60
80
100
120
140
160
1 mg / mL
0.5 mg / mL
0.3 mg / mL
0.1 mg / mL
A
/ mV s-1)1/2
2 4 6 8 10 12
J
/
cm
-2
10
20
30
40
50
60
R
2
= 0.995
R
2
= 0.9947
B
b
a
0.0 0.2 0.4 0.6 0.8 1.0 1.2
J
/
cm
-2
-20
0
20
40
60
10 mv s
-1
100 mv s
-1
A
At 20 mV/s in 100 µM Valacyclovir + 0.1 M
20 mL PBS (pH 7.4)
 Different concentration of GO -5 cycle
electrodeposition
Anodic peak current of 20
µM valacyclovir in 0.1 M
PBS (pH 7.2) -0.3 mg mL-1
(3, 5, 10 and 15 cycle)
CVs of different scan rate (A);
Plot of anodic response (a) AP and
(b) Valacyclovir

25
E / V ( Ag / AgCl)
0.6 0.7 0.8 0.9 1.0 1.1 1.2
J
/
cm
-2
0
20
40
60
80
0.6 0.7 0.8 0.9 1.0 1.1 1.2
0
10
20
30
40
Performance of rGO/GCE for Valacyclovir detection
CV response at 20 mV/s in 50 µM
Valacyclovir + 0.1 M 20 mL PBS (pH 7.4)
 rGO/GCE vs PBS
 Inset: bare GCE vs PBS
Concentration / 
0 10 20 30 40 50
J
/
A
cm
-2
0
10
20
30
40
R
2
= 0.992
R
2
= 0.985
B
E vs (Ag / AgCl) / V
0.7 0.8 0.9 1.0 1.1
J
/
cm
-2
10
20
30
40
50
10 nM
45.1 
A
0 10 20 30 40 50
J
/
A
cm
-2
0
10
20
30
40
R
2
= 0.992
R
2
= 0.985
B
0.7 0.8 0.9 1.0 1.1
J
/
cm
-2
10
20
30
40
50
10 nM
45.1 
A
Calibration plot of current response against
valacyclovir concentration.
DPV responses to the successive addition from 10
nM to 45µM

26
Simultaneous detection of acetaminophen and valacyclovir
E / V ( Ag/AgCl)
0.2 0.4 0.6 0.8 1.0
J
/
cm
-2
10
20
30
40
50 nM
45 
AP
Val
0
J
/
cm
-2
0
5
10
15
20
25
30
35
R
R
Performance of rGO/GCE for
simultaneous detection of 50µM
acetaminophen and valacyclovir. Inset:
bare GCE
Successive addition of 50 nM-45µM
E / V ( Ag / AgCl)
0.2 0.4 0.6 0.8 1.0 1.2
J
/
cm
-2
0
20
40
60
80
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0
10
20
30
40
A
AP
Val
Calibration curve of current response vs
concentration
E / V ( Ag/AgCl)
0.2 0.4 0.6 0.8 1.0
J
/
cm
-2
10
20
30
40
50 nM
45 
AP
Val
0 10 20 30 40 50
J
/
cm
-2
0
5
10
15
20
25
30
35
R
2
= 0.992
R
2
= 0.984
R
2
= 0.981
R
2
= 0.99
AP
Val

Interference, reproducibility and stability of developed sensor
27
(a) 25 µM AP and val
(b) 50 µM of each ascorbic
acid
(c) Dopamine
(d) Uric acid
(e) Glutathione
in 0.1 M PBS, pH 7.2
Relative anodic peak current response from (A)
̴2.5% for AP and 3.0% for Val-peak variation
Number of days
4 6 8 10 12 14 16 18 20 22
I
/
I
0
0
20
40
60
80
100
120
Stability test
Number of electrodes
1.0 2.0 3.0 4.0
J
/
cm
-2
0
2
4
6
8
10
12
14
Reproducibility test
0.0 0.2 0.4 0.6 0.8 1.0
J
/
cm
-2
a
b
c
d
e
50
f
Interference study
A
I
/
I
0
0
20
40
60
80
100
120 Acetaminophen
Valacyclovir
a b c d e f
B
0.0 0.2 0.4 0.6 0.8 1.0
J
e
f
B
Analysed through DPV
in 5 µM valacyclovir
A very low RSD of
1.08% (n=4)
5.26% current loss

28
Actual sample analysis in human plasma: simultaneous detection of
acetaminophen (325 mg) and valacyclovir (500 mg) generic tablets.
Added
(µM)
Found (µM) Recovery (%) RSD (%)
Acetaminophen Valacyclovir Acetaminophen Valacyclovir Acetaminophen Valacyclovir
5 5.3 4.96 106 99.33 2.17 5.3
10 10.1 9.43 101 94.33 5.5 0.99
15 14.25 14.55 95 97 1.75 2.75

29
Conclusion
 Graphene nanosheets (rGO) based
sensor has been developed for
simultaneous detection of AP and
valacyclovir
 A very low limit of detection
(LOD)-1.34 nM for valacyclovir;
simultaneous detection: LOD-4.65
nM for AP and 3.1nM for
valacyclovir
 Excellent stability, reproducibility
with no interference
 High recovery in real sample
analysis
 Highly suitable for pharmaceutical
formulation and bioavailability
testing
B.-R. Adhikari, M. Govindhan, H. Schraft, A. Chen. J.
Electroanal. Chem. 2016, 780: 241-248.

30
Exploring electrocatalytic activity of graphene based
nanocomposites with single walled carbon nanotubes (SWCNTs)
B.-R. Adhikari, M. Govindhan , A. Chen. Sensors 2015,
9:22490-22508
Synergistic catalytic behaviour of SWCNTs-rGO nanohybrid film

31
Cyclic voltammetric performance of Acetaminophen
50 µM acetaminophen, at 20 mV/s scan rate,
0.1 M PBS ( pH 7.2)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
I
/

-2
-1
0
1
2
rGO
0.0 0.1 0.2 0.3 0.4 0.5 0.6
I
/

-60
-40
-20
0
20
40
60
80
SWCNTs
0.0 0.1 0.2 0.3 0.4 0.5 0.6
I
/

-100
-50
0
50
100
150
SWCNTs-rGO
Drop casted 0.5 mg/mL SWCNTs and 4 mg/mL GO
on GCE; air dried and electrochemical reduction to
make SWCNTs-rGO nanohybrid thin film

32
Differential Pulse Voltammetric (DPV) performance of
Acetaminophen
0.1 0.2 0.3 0.4 0.5
I
/

0
2
4
6
8
80 M
5 M
rGO
E vs ( Ag AgCl) / V
0.1 0.2 0.3 0.4 0.5
I
/

100
150
200
250
300
350
400
5 nM
80 M
SWCNTs
0.1 0.2 0.3 0.4 0.5
I
/

100
150
200
250
300
350
400
5 nM
80 M
SWCNTs-rGO
0 20 40 60 80 100
I

0
1
2
3
4
5
6
7
0 20 40 60 80 100
I
/

0
50
100
150
200
250
300
0 20 40 60 80 100
I
/
50
100
150
200
250
300
350
SWCNTs-rGO>SWCNTs >rGO

33
Project 3: A High-performance Enzyme Entrapment Platform Facilitated by
a Cationic Polymer for the Efficient Electrochemical Sensing of Ethanol
Zn2+
Cys Cys
His
 Further explore biocompatibility properties of SWCNTs-rGO
nanohybrid
 Detailed study of enzyme entrapment platform for biosensor design

34
Biosensor fabrication
Graphite oxide
Graphene oxide ( 5
mg/mL) - ultrasonication
SWCNTs
SWCNTs (5 mg/mL) in
DMF- ultrasonication
Graphene nanocompsite-drop casted 2 µL each on GCE
cyclic voltammetry -0.6 to -1.5 V (5 cycles at 20 mVs-1) in
0.1M tris buffer solution- rGO/SWCNTs nanohybrid
Huang, N. M.; Lim, H. N.; Chia, C. H.; Yarmo, M. A.; Muhamad, M. R. Int. J.
Nanomed. 2011, 6, 3443.
MADQUAT 2 µL each for
ADH entrapment
Modified hummer’s method
Air dried

35
Surface characterization
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
A B
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
SEM images of (A) rGO, (B) SWCNTs and (C) SWCNT-rGO nanohybrids; (D) EDX
spectra of rGO (green), SWCNTs (blue) and SWCNTs-rGO nanobybrid (red).
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
A B
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
A B
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
A B
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
CV responses in a 0.1 M KCl
solution containing 2.5 mM
K3Fe(CN)6 at the scan rate of 20
mVs-1.

36
Wavenumbers ( cm-1)
1600 1620 1640 1660 1680 1700
Absorbance
0.00
0.01
0.02
0.03
0.04
Wavenumbers (cm-1)
1600 1620 1640 1660 1680 1700
Absorbance
0.00
0.01
0.02
0.03
0.04
Biocompatibility study on SWCNTs-rGO nanohybrid thin
film (ADH as model enzyme)
Free ADH ADH immobilized on poly-methyl
chloride(MADQUAT)
Linear association (r) = 0.92
Wavenumber (cm-1)
1000 1200 1400 1600 1800
b
a
(a) Free ADH (b) after
entrapment with polymer
Band assignment Band position Area %
ADH ADH+ Poly-
methylchloride
ADH ADH+ Poly-
methylchloride
Amino acid
absorption
1604,1614 1608,1614 13 10
ß-sheet 1633, 1689 1635 29 23
Random coils 1645 1646 25 23
ɑ-helix 1658 1658 22 21
ß-turns 1677 1675, 1686 11 14

37
Electrocatalytic behaviour of ADH onto SWCNT-rGO
nanohybrid for ethanol detection
CV responses (20 mVs-1); physisorbed ADH (green dashed line) in a 0.1M tris
buffer containing 50 mM ethanol + 10 mM NAD+ and only 10 mM NAD+(blue
dashed line).
0.0 0.2 0.4 0.6
I
/

-40
-20
0
20
40
60 D
I/
µA
0.0 0.2 0.4 0.6
I
/

-20
0
20
40
60
A
I/
µA
0.0 0.2 0.4 0.6
I
/

-20
0
20
40
60
B
I/
µA
0.0 0.2 0.4 0.6
I
/
A
-20
0
20
40
60 C
I/
µA
ADH-rGO ADH-SWCNTs
ADH-SWCNTs-rGO
SWCNTs-rGO
10 mM NADH

38
Optimization of proposed biosensor (ADH-SWCNTs-rGO/GCE)
pH effect
7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
I
/

0.01
0.02
0.03
0.04
0.05
B
MADQUAT concentration ( mg mL-1)
0 10 20 30 40 50 60 70
I
/

0.00
0.01
0.02
0.03
0.04
0.05
0.06
A
 On 20 µM ethanol
 50 mg/mL MADQUAT
concentration
 pH 8.2
(A) CVs at scan rates of 20, 40, 50, 60 and 70
mVs-1.
(B) Plot of anodic peak currents versus the scan
rates obtained from A (surface controlled process)

39
Time / Sec
1000 1200 1400 1600 1800 2000 2200
0.8
1.0
1.2
1.4
1.6
1.8
2.0
5 M
100 M
C
I/
µA
Concentration/ M
0 200 400 600 800
1.0
1.2
1.4
1.6
1.8
2.0
R
2
= 0.998
D
I/
µA
R
2
= 0.99
Concentration / mM
0 5 10 15 20 25 30 35
0
2
4
6
8
10
R
2
= 0.986
B
I/
µA
-0.2 0.0 0.2 0.4 0.6
I
/
A
-40
-20
0
20
40
60
1 mM
30 mM A
Analytical performance of biosensor (ADH-SWCNT-rGO/GCE)
(A) CV responses at 20 mVs-1 (1 – 30 mM ethanol)
(B) Calibration plot of the current responses derived from A
(C)Amperometric responses from 5 - 800 µM (Eapp= 0.5V)
(D) Calibration plot of the current responses derived from C.
0.1M tris buffer (pH 8.2)+ 10 mM NAD+.

40
Time / Sec
600 700 800 900 1000
I
/

0.50
0.55
0.60
0.65
0.70
0.75
a
b c d e
f
B
Time (s)
1000 2000 3000 4000
I
/
I
0
0
20
40
60
80
100
120
Sample Concentration
added (mM)
Concentration
detected (mM)
Recovery (%) RSD (%)
10.00 9.30 93.0 3.3
Wine 20.00 19.80 98.9 2.4
30.00 29.91 99.7 4.1
10.00 9.82 98.2 1.9
Beer 20.00 20.06 100.3 3.6
30.00 29.55 98.5 2.1
10.00 10.40 104.0 4.3
Blood alcohol 20.00 20.30 102.0 4.7
30.00 31.00 105.0 1.6
Interference, stability and real sample analysis of proposed biosensor
Time / Sec
600 700 800 900 1000
I
/

0.50
0.55
0.60
0.65
0.70
0.75
a
b c d e
f
B
Time (s)
1000 2000 3000 4000
0
20
Interference tests- 20 µM ethanol (a),1mM of
each ascorbic acid (b), glutathione (c), glucose
(d), uric acid (e) and 20 µM ethanol (f)
10 mM NAD+ in 0.1 M tris buffer.
Stability test: in 20 µM ethanol
Real sample analysis
Eapp: 0.5 V

41
Conclusion
 Studied biocompatibility behaviour of
SWCNTs-rGO nanohybrid (no alteration in
structure)
MADQUAT entrapped ADH biosensor on
SWCNTs-rGO nanohybrid for ethanol
detection
Different carbon based platforms have
been studied.
The synergistic enhancement of
SWCNTs-rGO nanohybrid has been
revealed with superior activity
B.-R. Adhikari, H. Schraft, A. Chen. Analyst 2017,
142:2595-2602

42
Project 4: Integrated Bifunctional Electrochemcial Approach
for Efficient Bacterial Disinfection
Live E. coli cells
Bifunctional
After 10 minutes
 Attempt to broadening the application of nanostructured materials
 A new approach of combining two nanostructred materials (bifunctional)-
photocatalyst (nanoporous TiO2) and electrocatalyst (RuO2) for highly
synergistic activity for electrochemical water disinfection

43
Fabrication of bifunctional electrode
Ti plate (1.25 cm x 0.8 cm
x 0.5 mm)-anode
Pt coil-cathode
1st anodization
0.3 wt% ammonium fluoride;
NH4F and 2wt% water in
ethylene glycol Eapp 50 V; 5hrs
Rough nanoporous-
removed by masking
tape
Rough nanoporous-
removed by masking
tape
2nd anodization;
2 hrs
Rutile nanoporous TiO2
3rd anodization;
15 min
Anatase nanoporous TiO2
450 0C for 4 hrs
in oven
Working nanoporous TiO2
Electrochemical
reduction
- 5 mA cm-2 for 10
min; 0.1M H2SO4
Ruthenium (III) chloride
hydrate (RuCl3.x H2O) Calcination; 450
oC for 2 h
RuO2
Bifunctional electrode
Electrochemical bacterial disinfection through amperommetry; Eapp 1.2 V; 100
mL of 0.05 Na2SO4

44
Energy (KeV)
Intensity
(a.u.)
Nanoporous TiO2
RuO2
D
Energy (KeV)
0 1 2 3 4
Intensity
(a.u.)
C
TiO
Ru O
Ru
RuO2
Nanoporous TiO2
C
Time (min)
50 100 150 200
j
(mA
cm
-2
)
0
5
10
15
20
25
TiO2/Ti
RuO2/Ti
D TiO2
/Ti/RuO2
Characterization of the bifunctional TiO2/Ti/RuO2 electrode

45
Time / min
0 5 10 15 20 25 30 35
ln
(C/C
0
)
-15
-10
-5
0
5
TiO2/Ti/RuO2
TiO2/Ti
RuO2/Ti
C
Time / min
10 20 30 40 50 60
Log
10
reducti
0
2
4
6
8
RuO2/Ti
TiO2/Ti
TiO2/Ti/RuO2
Control
A
Time / min
10 20 30 40 50 60
Log
10
reduction
0
2
4
6
8
10 B
a
b
c
d
e
RuO2/Ti
RuO2/Ti
Time(min)
Time(min)
Time(min)
TiO2/Ti/RuO2
Time (min)
0 5 10 15 20 25 30 35
ln
(C/C
0
)
-15
-10
-5
0
5
TiO2/Ti/RuO2
TiO2/Ti
RuO2/Ti
C
5
Time / min
10 20 30 40 50 60
Log
10
reduction
0
2
4
6
8
10
RuO2/Ti
TiO2/Ti
TiO2/Ti/RuO2
Control
A
Time / min
10 20 30 40 50 60
Log
10
reduction
0
2
4
6
8
10 B
a
b
c
d
e
TiO2/Ti/RuO2
RuO2/Ti
Time(min)
Time(min)
5
5 min 10 min 15 min
30 min 25 min 20 min
5 min 10 min 15 min
5 min 10 min 15 min
5 min 10 min 15 min
5 min 10 min 15 min
5 min 10 min 15 min
Performance of electrodes for bacterial disinfection
(initial count 2.3 x 108 CFU / mL)
5
Time / min
10 20 30 40 50 60
Log
10
reduction
0
2
4
6
8
10
RuO2/Ti
TiO2/Ti
TiO2/Ti/RuO2
Control
A
Time / min
10 20 30 40 50 60
Log
10
reduction
0
2
4
6
8
10 B
a
b
c
d
e
TiO2/Ti/RuO2
RuO2/Ti
Time(min)
Time(min)
5
TiO2/Ti
(B) ROSs scavenger
experiments in bifunctional : no
scavenging (a), 10 mM of each
sodium azide (b) mannitol (c),
sodium pyruvate (d), sodium
thiosulfate (e); ( major ROS
H2O2)
Culturable cell reduction
(C) Disinfection kinetics: 0.62
min-1 ( TiO2/Ti/RuO2); 0.28
min-1 (TiO2/Ti); 0.14 min-
1(RuO2 /Ti)

46
Time(min)
Time(min)
Time(min)
Time(min)
Time(min)
Time(min)
Bacterial cell viability estimation
LIVE/DEAD® BacLight™ stain through confocal scanning laser
microscopy (A) 0 min; (B) 5 min; (C) 30 min; (D) Biovolume count
SEM analysis (E) 0 min; (F) 30 min
Biomolecule leakage (A) TOC (B)
Protein concentration
The LOD of spread plate method is ˂ 100 CFU/mL for 1/10 dilutions

47
Time of
treatment
(minutes)
Nutrient broth (well) Nutrient broth enriched with 30 mM
sodium pyruvate (well)
Average MPN Standard error (n=3) Average MPN Standard error (n=3)
30 210 6.3 480 6.3
40 8.6 4.1 18.2 4.1
50 0.66 - 5.4 1.6
60 0 - 0 -
70 0 - 0 -
Viable but non culturable (VBNC) state after bifunctional treatment
A
Resuscitated
B
Most probable number (MPN) of E. coli calculated
through American Public Health Association

48
Variables Initial 10 minutes 20 minutes 30 minutes 40 minutes 50 minutes
Initial 1 -0.037 -0.363 -0.439 -0.245 -0.340
10 minutes -0.037 1 0.368 -0.197 -0.144 -0.299
20 minutes -0.363 0.368 1 -0.109 -0.123 -0.248
30 minutes -0.439 -0.197 -0.109 1 0.668 0.162
40 minutes -0.245 -0.144 -0.123 0.668 1 0.325
50 minutes -0.340 -0.299 -0.248 0.162 0.325 1
Metabolomics study through NMR
1H NMR processed through spin works
All the NMR spectra analyses for Principle component
analysis (PCA) correlation matrix- XLSTAT version
2016.5 software

49
• A total of 106 metabolites.
• A total of 38 primary metabolites -
Initial sample
• Major metabolite loss-30 min of
electrochemical treatment,
• 17 vital metabolites lost.
• The lack of key metabolites for
TCA cycle, DNA synthesis,
lipopolysaccharide and
peptidoglycan synthesis- induce
cell death- confirmed by SPM in
NA
The NMR spectra positions used to
search through the Escherichia coli
Metabolome Database (ECMDB,
http://www.ecmdb.ca) for potential
metabolites and verified through
the freely available Biocyc
(http://biocyc.org) database
Confirmation of metabolites through ECMDB database

50
Conclusion
Synergistic behaviour of photocatalyst (TiO2) and electrocatalyst (RuO2) in
one electrode ( bifunctional) have been studied
 Very efficient bacterial disinfection through the bifunctional electrode in
comparision to their individual photocatalyst (TiO2) and electrocatalyst (RuO2)
counterparts
 A high disinfection rate (0.62 min-1) with >99.999% of bacterial removal
within 20 min throughTiO2/Ti/RuO2 bifunctional electrode
 Very low power consumption (1.2V) and evironmemental friendly
technology
 No VBNC state of bacteria found for longer time ( nil within 50 min)
 Studied bacterial mtabolomics of different treated sample with strongly
relates to mass death of E. coli after 30 min of bifunctional treatment
This chapter has been submitted to Water Research (a high impact peer reviewed
journal)

51
Future prospectives
 A combining approach of nanomaterials have great synergistic effect. This
approache can be utilized further for the different electrochemical processes
Further study the synergistic nature of SWCNTs-rGO nanohybrid thin film
for biosensing application through the indepth study of enzyme-substrate
catalysis
Functionalization of these nanohybrid film may further improve the
biocatalytic performance by the formation of stable covalent bond
 Doping with conducting metal nanoparticles on TiO2 nanoporous and then
combining it with electroactive catalyst may further improve the
performance with out using UV- visible light
By reducing band gap of photocatalyst, the performance can be improved in
visible light which my be useful not only for energy efficient bacterial
disinfection but also for electrochemical biosensing application

52
Acknowledgement
 Dr. Aicheng Chen (supervisor)
 Dr Heidi Schraft (Co-supervisor)
 Dr Neelam Khaper (Committee
member)
 Dr Antonella Badia (External
examiner)
 Michale moore and biology lab
members
 Chemistry department
 Instrumentation lab members
 Dr. Chen’s group members
NSERC for PGSD during PhD

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