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Nanomaterials Based Electrochemical Approaches for
Biosensing and Bacterial Disinfection
Bal Ram Adhikari
PhD candidate Biotechnology
1
oAdvisor: Dr. Aicheng Chen
Advisor: Dr. Aicheng Chen
oCo-advisor: Dr. Heidi Schraft
Co-advisor: Dr. Heidi Schraft
oCommittee member: Dr. Neelam Khaper
Committee member: Dr. Neelam Khaper
oExternal examiner: Dr. Antonella Badia
External examiner: Dr. Antonella Badia
oCommittee chair: Dr. Wely Floriano
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
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Background and Rational of thesis
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Rationale of thesis
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12
Research objectives
Objective 1: Study the synthesis, characterization and optimization of
carbon based nanomaterials for electrochemical sensing/biosensing
Objective 2JStudy the preparation and analytical performance of reduced
graphene oxide (rGO) towards detection of acetaminophen
Objective 3JOptimize 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
2+7 00
*
(
13
Electrochemical Methods
2? " 2
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
C4
2
?8
56!+/-8AC
%C4
K
!'
C 16
(#(
#
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
&2?'56!+/>'&5
>?8
65*"
B
5? "#
'
56!+/>'
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
0.2M
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
R2L 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
&5?'56!+/M'
'##
4#6%"*
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
C #
# "
?-
'
>'=N'
#"
? "
E
4 B.-R. Adhikari 'C" &2. Electrochim. Acta
2015 0J.=
22
Project 2: Simultaneous and Sensitive Detection of Acetaminophen and
Valacyclovir Based on Two Dimensional Graphene Nanosheets
?" 4J =
4"""#4
23
Electrode fabrication: Methodology
-8A56!+.
Raman shift ( cm
-1
)
800 1000 1200 1400 1600 1800
In ten si ty
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
?" #
(#
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
E vs ( Ag / AgCl) / V
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
&*
µ'""'
56!+/-A
- > >
2?&@5
&5 #
?"
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
R2 = 0.992
R2 = 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
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
2#
""
5?"
'>N'
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
5 C8C2
>N'
""7J
#C2
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
Concentration /
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
%"*A)
OP>Q&5-Q?*"
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
E vs ( Ag / AgCl) / V
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 / I0
0
20
40
60
80
100
120
Acetaminophen
Valacyclovir
a b c d e f
B
B
&5?
5 µM valacyclovir
&"%!
=QL
>0Q
28
Actual sample analysis in human plasma: simultaneous detection of
acetaminophen (325 mg) and valacyclovir (500 mg) generic tablets.
&
N'
3N'
%"Q
%!Q
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
C C #
# "
&5
""
& "
A- ' ""@
J A0>
' &5 -'
""
4 # #
+ "
+ #
#"#
B.-R. Adhikari 'C" +! &2J.
Electroanal. Chem.0 /=J=
30
Exploring electrocatalytic activity of graphene based
nanocomposites with single walled carbon nanotubes (SWCNTs)
B.-R. Adhikari ' C" & 2. Sensors2015
.J.>=
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)
E vs ( Ag / AgCl) / V
0.0 0.1 0.2 0.3 0.4 0.5 0.6
I /
-2
-1
0
1
2
rGO
E vs ( Ag / AgCl) / V
0.0 0.1 0.2 0.3 0.4 0.5 0.6
I /
-60
-40
-20
0
20
40
60
80
SWCNTs
E vs ( Ag / AgCl) / V
0.0 0.1 0.2 0.3 0.4 0.5 0.6
I /
-100
-50
0
50
100
150
SWCNTs-rGO
>8A!,2)(8A
CC2@
*!,2)(C#
32
Differential Pulse Voltammetric (DPV) performance of
Acetaminophen
E vs ( Ag / AgCl) / V
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
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-rGO
Concentration /
0 20 40 60 80 100
I
0
1
2
3
4
5
6
7
Concentration /
0 20 40 60 80 100
I /
0
50
100
150
200
250
300
Concentration /
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
Zn
2+
Cys Cys
His
Further explore biocompatibility properties of SWCNTs-rGO
nanohybrid
Detailed study of enzyme entrapment platform for biosensor design
34
Biosensor fabrication
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
'R
&
35
Surface characterization
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
AB
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
!'&C 6!,2)(2!,2)(C#@K
C !,2)(#!,2)(C##
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
AB
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
AB
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
Energy, keV
0.5 1.0 1.5 2.0
Intensity
Oxygen
Carbon
D
AB
C
A B
C
SWCNTs-rGO
SWCNTs
rGO
2? ' <2
> '
<-32)0
?
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
3&+#
Band assignment Band position Area %
ADH ADH+ Poly-
methyl chloride
ADH ADH+ Poly-
methyl chloride
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).
E vs (Ag / AgCl) / V
0.0 0.2 0.4 0.6
I /
-40
-20
0
20
40
60
D
I/ µA
E vs ( Ag / AgCl) / V
0.0 0.2 0.4 0.6
I /
-20
0
20
40
60
A
I/ µA
E vs ( Ag / AgCl) / V
0.0 0.2 0.4 0.6
I /
-20
0
20
40
60
B
I/ µA
E vs (Ag / AgCl) / V
0.0 0.2 0.4 0.6
I / A
-20
0
20
40
60
C
I/ µA
&+C &+!,2)(
&+!,2)(C
!,2)(C ')&+
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
N'
>8A'&1E&(
+=
& 2? > 0 /
?
6 5 * "
#&
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
E vs (Ag / AgCl) / V
-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)
&2??I-'
62#"&
2&>=N'L>?
2#"2
'#+=M')&M
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 (%)
.-
.-
--
Wine
.=
.=.
-
..
../
.=
.=
.
Beer
0
-
-0
-
.>>
.=>
-
Blood alcohol
-
/
-
-
>
0
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
I / I
0
0
20
40
60
80
100
120
7µ' '
##
µ'
')&M'#
!#Jµ'
Real sample analysis
J>?
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 + ! & 2. Analyst2017
J>.>0
42
Project 4: Integrated Bifunctional Electrochemcial Approach
for Efficient Bacterial Disinfection
Time (min)
Live E. coli cells
Bifunctio nal
After 10 minutes
&#
& # #
( %
"
43
Fabrication of bifunctional electrode
(>4=
4>
5
$
0.3 wt% ammonium fluoride;
NH4F and 2wt% water in
ethylene glycol >?@>
Rough nanoporous-
removed by masking
tape
Rough nanoporous-
removed by masking
tape
$@
Rutile nanoporous TiO2
-$@
>
Anatase nanoporous TiO2
>2
"
Working nanoporous TiO2
>&
@'+!
Ruthenium (III) chloride
hydrate (RuCl3.x H2O) 2@>
2
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 TiO
2
RuO
2
D
Energy (KeV)
0 1 2 3 4
Intensity (a.u.)
C
TiO
Ru O
Ru
RuO
2
Nanoporous TiO
2
C
Time (min)
50 100 150 200
j (mA cm
-2
)
0
5
10
15
20
25
TiO
2
/Ti
RuO
2
/Ti
D
TiO
2
/Ti/RuO
2
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 reduction
0
2
4
6
8
10
B
a
b
c
d
e
TiO
2
/Ti/RuO
2
RuO
2
/Ti
RuO
2
/Ti
Time (min)
Time (min)
TiO
2
/Ti/RuO
2
Time (min)
0 5 10 15 20 25 30 35
ln (C/C
0
)
-15
-10
-5
0
5
TiO
2
/Ti/RuO
2
TiO
2
/Ti
RuO
2
/Ti
C
Time / min
10 20 30 40 50 60
Log10 reduction
0
2
4
6
8
10
RuO2/Ti
TiO2/Ti
TiO2/Ti/RuO2
Control
A
A
Time / min
10 20 30 40 50 60
Log10 reduction
0
2
4
6
8
10
B
a
b
c
d
e
TiO
2
/Ti/RuO
2
RuO
2
/Ti
RuO
2
/Ti
Time (min)
TiO
2
/Ti/RuO
2
Time (min)
0 5 10 15 20 25 30 35
ln (C/C
0
)
-15
-10
-5
0
5
TiO
2
/Ti/RuO
2
TiO
2
/Ti
RuO
2
/Ti
C
> >
- >
> >
- >
> >
- >
> >
- >
> >
- >
> >
- >
Performance of electrodes for bacterial disinfection
(initial count 2.3 x 108 CFU / mL)
Time / min
10 20 30 40 50 60
Log10 reduction
0
2
4
6
8
10
RuO2/Ti
TiO2/Ti
TiO2/Ti/RuO2
Control
A
A
TiO
2
/Ti/RuO
2
RuO
2
/Ti
RuO
2
/Ti
Time (min)
TiO
2
/Ti/RuO
2
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)
2#
(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)
Bacterial cell viability estimation
LIVE/DEAD® BacLight™ stain through confocal scanning laser
microscopy &@6>@2-@6"
SEM analysis @3-
Biomolecule leakage &(26
5
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 -/ -0- -. > -
10 minutes -/ -0= ./ ..
20 minutes -0- -0= . - =
30 minutes -. ./ . 00= 0
40 minutes > - 00= ->
50 minutes - .. = 0 ->
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
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•& -= #
7
•'9 # -
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•( * * #
(2& )&
# !5'
)&
()'%
Escherichia coli
'##2'6
J88#
#"
"#6
J88##
Confirmation of metabolites through ECMDB database
50
Conclusion
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This chapter has been submitted to Water Research (a high impact peer reviewed
journal)
51
Future prospectives
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52
Acknowledgement
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+!2"
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