COMSATS Institute of Information Technology Islamabad,
Pakistan.
Electrochemical Studies of Nanostructured
Protein Based Immunosensors.
By
Saima Rafique
Supervised by
Prof. Arshad Saleem Bhatti
Center for Micro and Nano devices,
Dept. of Physics, COMSATS Institute of Information
Technology, Islamabad.
Collaborators
Prof. Chang Mang Li
School of Chemical and Biomedical Engineering,
Nanyang Technological University, Singapore.
1
Outline
●
●
●
●
●
Introduction
Cancer
Cancer biomarkers
Immunosensors
Importance of Supporting matrix for immunosensor
Section II
Section I
(Early stage cancer diagnostics using cancer
(Growth kinetics and adhesion
biomarkers)
behavior of self assembled
PART I (Colon cancer)
monolayers (SAM))



Steps for preparation
Surface morphology (AFM)
Growth kinetics
 f-d Curves
 Conclusions
Acknowledgements
 Step of preparation of an immunosensor
 Surface morphology (FESEM & AFM)
 Cyclic Voltammetry
 Limit of detection
 Specificity and stability of immunosensor
 Conclusions
PART II (Prostate cancer)





Sandwich immunosensor preparation
Morphology of Au and silica NPs (FESEM)
Differential pulse voltammetry
Nyquist plot for resistance
2
Conclusions
Motivation
How frequently
people go for
regular medical
check up
Ϯ
Cancer statistics in Pakistan
*Cancer statistics in Pakistan
Breast
Lungs
Blood
6%
Cancer control
Primary prevention
• Public information and
education
• Self examination
Secondary prevention
• Early diagnosis
• Screening and therapy
Ϯ N. Bano et al, Asian J Pharm Clin Res 6, (2013), 13-17.
*A. Gul et al, J. Med. Sci. 20, (2012), 67-70.
ǁM. Hanif et al, Asian Pac. J. Cancer. Pre. 10, (2009), 227-230.
8%
12%
13%
4%
3%
Prostate
22%
18%
14%
Colon
Liver
Brain
Others
Bone
In Pakistan most common cancers
are
•
•
•
•
Breast Cancer (22% of all cases)
Lung cancer (18%)
Prostate cancer (13%)
Colon (8%)
3
Cancer: An introduction
Cancer
biomarkers

Disease
caused by an uncontrolled growth of abnormal cells in a
part of the body
Different types of cancer
• A cancer biomarker refers to a
biomarkers
substance that is indicative of the
*
presence of cancer in the body
• α-Fetoprotein (Liver cancers)
• Cancer antigen-125 (Ovarian cancer)
• Prostate specific antigen (Prostate cancer)
• Carcinoembryonic antigen (Colon cancer)
Prostate cancer
• Prostate cancer is a disease in which
cells in the prostate gland
start to grow
uncontrollably,
forming tumors
Colon cancer
• Colon cancer, is a cancer from
uncontrolled cell growth in
the colon (parts of the large intestine)
*A. Mishra and M. Verma, Cancers, 2, (2010), 190-20.
4
Cancer detection: Enzyme-linked
immunosorbent assay (ELISA)
Disadvantages
 The ELISA has been used as a diagnostic tool in medicine
 Enzyme reaction is short term so signal must be read as soon as possible.
 Different types of ELISA are
 It being an enzymatic reaction, even small quantities of non-specific
• binding might result in false signal.
•
•
Direct assays
Indirect
assays
ELISAs
is quite
complex,
washing.
Sandwich assays
including multiple steps of incubation and
Antibody (Ab)
*
Antigen (Ag)
Fig: ELISA
*M. Thompson, Anal. Meth., 45, (2010), 1757-1759.
5
Label free Immunosensor
 Immunosensors are biosensors based on specific antigenantibody interactions
It eliminates the need for tags
Simplifying assay designed
Fast detection
Fig: Schematics of immunosensor
• Current
• Impedance
Supporting Matrix
Antibody
*A. Sharma , Z. Matharu, G. Sumana, Thin Solid Films, 38, (2010), 245-251
Antigen
6
Importance of supporting matrix for
immunosensor
 Supporting matrix are important because
•
•
•
Sensitivity
Specificity
Reduces physical adsorption
 Different types of supporting matrix used are
•
•
•
•
•
Self assembled monolayer
Carbon nanotubes
Polymers brushes
Gold nanoparticles
Silica nanoparticles
Self assembled monolayers
Polymer brushes
Gold NPs
Carbon nanotunes
Fig: Various types of supporting matrix
7
Supporting matrix: Self assembled
monolayer (SAM)
•SAM are organic layers formed on a
solid substrate by spontaneous
organization of molecule*.
•SAM is formed by the strong chemical
interaction between the substrate and
Fig: Schematics of SAM
head group of selected organic molecule.
Advantage of using SAM is it reduces physical adsorption of biomolecules.
*Christopher. J. Love, L. A. E, Chem. Rev, 105 (2005) 1103-1169
8
Supporting matrix: Gold nanoparticles

The different nanomaterials are developed, among them
gold nanoparticles (AuNPs) has been frequently used
because of
•
●
Easy functionalization
Good biocompatibility
They provide high surface area, more no of biomolecules can be attached.
Fig: Various shapes of gold nanostructures
*S. Barua, J. Yoo and S. Mitragotri, Proc. Natl. Acad. Sci., 110, 3270 (2013).
9
Supporting matrix: Polymer
brushes

Polymer brush is a layer of polymers attached with one
end to a surface.

Homopolymer (a)

Copolymer
Homo polymer brushes
Graft copolymers (e)
(A-A-A-A-A-A-A-A-A-A-A-A-A-A-A)
B-B-B-B
Alternating (b)
(BABABABA)
Random (c)
(BBBABBBA
BA)
Block (d)
(BBBAAABB
BAAA)
B-B-B-B
(a) Polytetrafluoroethylene (PTFE)
(b) Poly(methyl methacrylate) and poly(Nisopropylacrylamide) [poly(PMMA-alt-PNIPAM)]
(c) poly(n-octadecyl methacrylate)-b-poly(t-Bu acrylate)
[(pODMA-b-ptBA]
(d) Poly (styrene-block-methtyl (methacrylate) [PS-b-PMMA]
(e) Poly[oligo(ethylene glycol) methacrylate-co-glycidyl
methacrylate] (POEGMA-co--GMA)
High density of polymer brushes reduces the physical adsorption.
10
Aim of Study
 The aim of the present research work is to
●
Study the growth kinetics and adhesion parameters of
assembled monolayers
self
Diagnose cancer with improved
●
●
●
●
Sensitivity
Specificity
Limit of detection (LOD)
12
Section I
Growth kinetics and adhesion characteristics of self
assembled monolayer by force spectroscopy
13
Experiments
Si Substrate with
10nm Cr/100nm Au
Annealed at 300°C for 3 hr
Washed and rinsed with ethanol
Washed + Dried
Chip incubation in SAM*+ Ethanol
Solution (1, 3, 5, 7, 9hrs)
Annealing SAM at 100°C for 1hr
*SAM= 16- Mercapto-1-hexadecanol
• Two sets of samples were prepared one as grown and other is annealed
14
AFM analysis
 The thickness of as grown SAM
increased from 9 ± 1nm to 56 ± 3nm as
incubation time increased from 1 to 9 hrs.
 In case of annealed SAM the thickness of
monolayer changes from 5 ± 1 nm to 13 ±
1nm.
Annealing improves the thickness and
surface morphology of SAM.
As grown SAM
t= 1, 5 ,9hrs
Annealed SAM
t= 1, 5, 9 hrs
15
Conti….
60
(a) As - grown
24
50
 In case of annealed SAM, the thickness
dropped significantly and doubled the
surface coverage.
21
40
30
18
20
10
15
50
20
(b) Annealed
40
10
35
5
30
0
In case of annealed SAM, the thickness of
monolayer for 1 hr was about 5 ± 1 nm,
more or less the height of a single monolayer.
45
15
Surface coverge (%)
coverage
saturates to 22% after 3hrs of incubation.
Thickness (nm)
As the thickness increased the surface
1
2
3
4
5
6
7
8
9
25
10
Incubation time (hr)
Fig: Thickness and surface coverage verses
incubation time
16
Aspect ratio

The aspect ratio is

For as grown SAM the aspect ratio
increased due to the increase in thickness
with the incubation time.

Average (Thickness/surface coverage)
3.0
2.5
1.5
1.0
Annealed
0.5
0.0
For annealed SAM it reduced significantly
due to decreased in thickness as well
As-grown
2.0
0
2
4
6
8
10
Incubation Time (hrs)
Fig: Aspect ratio verses time
increased in surface coverage.
The aspect ratio improved by almost 90% from the as – grown to the annealed
SAM for the sample incubated for 9 hrs.
17
Growth kinetics
 (a) SAM assembling on the surface
170
 (b) The adsorbed SAM acts as a
increased
 (c) Coalescence of molecules increased
Average grain size (nm)
nucleation sites so multilayer formation
 In case of annealed SAM, as incubation
time increased the relative change in
density decreased which showed that
N= 45
160
(c)
150
As grown SAM
140
130
(b)
N= 93
120
N= 82
N= 60
(a)
110
N= 74
100
Annealed SAM
N= 303
N= 210
N= 295
90
0
2
4
N= 170
N= 174
6
8
10
Incubation time (hour)
Fig: Growth kinetics of islands
molecules diffused as a larger grain on
adsorption site
For as – grown SAM, the number of islands decreased and their average size
18
increased with the increased incubation time.
Atomic force microscopy (AFM)
 AFM is a technique for analyzing the surface
morphology of different materials
 The tip scan over the surface
 The laser beam deflected from cantilever was
detected by photodiode
 At A, the cantilever is far from the surface
(no interaction with the surface).
Fig: Atomic force microscopy
 At B, it approach toward the surface, the
tip interacts with the sample and a jump in
contact occurs
 At C, embed in the surface
 At D, move away towards the surface
 At E, Back to normal position
*M. Brogly, O. Noel, H. Awada, G. Castelein and J. Schultz, C. R. Chimie ,9 (2006), 99–110
Fig: Force distance spectroscopy
19
F-d curves
As Grown
100
Annealed
(a) 1 hour
8
50
4
0
-4
-50
• It shows the variation in

loop energy
slope of the loop

pull off/ adhesion force
Force (nN)
100
-8
100
(b) 5 hours
50
50
0
0
-50
-50
-100
150
-100
(c) 9 hours
100
100
50
50
0
0
-50
-50
-100
-100
-150
0
100
200
300
0
100
200
-150
300
400
Distance (nm)
Fig: F-d cures by AFM
The morphology of SAM effects the force distance curves.
20
Force (nm)
• The figure shows the f-d curves for 1, 5, 9 hrs.
0
Adhesion properties of SAM
 The pull off force is the force required to pull off
 Loop energy was calculated by the loop area of the
f-d curve.
 As more molecules come under the tip requires
more
energy to overcome the adhesive force so there
was
variation in loop energy.
(a)
Pull off force (nN)
(c)
80
As grown
60
(b)
40
20
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Aspect ratio
3.0
2.5
-14
 (a) Initially, the SAM was assembling or lying on
the surface (b) As agglomeration increased it
approached to a limiting value of 38 nN (c) Further,
coalescence of molecules increased the pull off force
Annealed
100
Loop Energy(10 J)
the tip from the surface
120
Annealed
2.0
As - grown
1.5
1.0
0.5
0.0
0.0
0.5
1.0
1.5
2.0
2.5
Aspect ratio
Fig: Loop energy and pull off force
verses aspect ratio
The increase in the pull off force and loop energy with increasing aspect ratio is a clear
21
indication of the agglomeration of molecules on the surface.
3.0
Calculation of elastic modulus

Two models were mostly used
●
●
●
JKR model (Johnson-Kendall-Robert) *
DMT model (Derjaguin-Mullar-Toporov) Ϯ
Difference between the JKR and DMT models occurs in assuming the
nature of forces acting between the tip and the substrate

Maugisǁ analyzed both the JKR and DMT models and suggested that the
transition between these models by dimensionless parameter
Where
Zo= equilibrium separation between tip and sample
R= Tip radius
K= Elastic modulus of tip
WA= Work of adhesion
*R. W. Carpick et al, J. Colloid. Inter. Sci., 211, (1999), 395–400.
ϮO. D. S. Ferreira et al, Applied Surface Science, 257 (2010), 48-54.
ǁJ. P. Aim et al, J. Appl. Phys. 76, (1994), 754-762.
22
Conti….
•If λ>5 the JKR model applies
•If λ<0.1 the DMT model applies
•Values between 0.1 and 5 correspond to the transition regime
•In JKR theory the interfacial energy or work of adhesion is given by
Where F = Pull off force,
R = Tip radius
•While the contact area is given by
J. Drelich, G. W. Tormoen, J. Colloid and Interface Science, 280 (2004), 484-491.
23
Conti….
Where P = applied load
R = Radius of the tip
W = work of adhesion
K=
E, ν elastic modulus and Poisson ratio of substrate
•
Using these values the contact area was 4 nm2
•
The effective modulus of the substrate and tip is given by
Where E, Ei and ν, νi are the elastic modulus and Poissons ratio of sample and the
tip
•
The average value of elastic modulus came out to be 0.3, 1.3 GPa for as
grown
and annealed SAM respectively.
24
Size dependence of elastic modulus
4
upon the particle size as well as thickness
of the layers.
 For annealed SAM it reached to the value
Annealed
Elastic modulous (GPa)
 The elastic modulus essentially depends
3
2
1
0
0.0
of 3.3 GPa which give rise the step difference
of 1 GPa between as grown and annealed
As - grown
0.5
1.0
1.5
2.0
2.5
3.0
Aspect ratio
Fig: Elastic modulus verses aspect ratio
SAM.
As the particle size decreased the molecules under the tip deformed
more easily give rise an increase in elastic modulus.
25
Conclusions (Section I)
•The kinetics of SAM formation was studied
•From the growth of island size it is cleared that initially it
grows as multilayered structure and then it agglomerates.
• The reconstruction take place onto the surface by annealing
which has significant effect on island area as well as on island
density
• The adhesive and elastic properties showed dependence on
the
growth stages and vary with the size of the island
26
Section II
Part I
Comparative study of label-free electrochemical
immunoassay on various gold nanostructured
electrode
Collaborators
Prof. Chang Mang Li, Dr. Gao Chuxian
School of Chemical and Biomedical Engineering, Nanyang Technological
University, Singapore.
27
Schematics of immunosensor
preparation
Carcinoembryonic antibody (CEA)
Nanstructures
Hydrogen
tetrachloroaurate
HAuClO4 ( mM)
Perchloric acid
HCLO4 (M)
Voltage (V)
Time(min)
Pyramid
40
0.1
-0.08
2
Spherical
40
0.1
-0.2
2
Rod
4
0.1
-0.08
2
28
Surface morphology
The average edge length of the
pyramid nanostructures was 205 ± 2nm.

 The spherical nanostructure had an
average diameter of 15 ± 1 nm.
 Where as nano rods had an average
diameter of about 120 ± 1 nm.
Nanostructures
Surface
Surface
area (µm2) coverage %
Pyramid
45.5
52.9
Spherical
52.1
63.9
Rod
42.3
40.7
The spherical nanostructures smaller in
size have high surface area and coverage.
Fig: FESEM and AFM images of pyramid,
spherical and rod like nanostructures. 29
Electrochemical impedance
technique
Control
EIS data analysis
 Impedance methods involves a small
V
I
PS
amplitude
Equivalent
circuit model
sinusoidal
signal to system under
Cyclic
voltammetry
 Most common used model
is
investigation and measure
the
impedance, current
Reference
Rct
Differential pulse voltammetry
Counter

In cyclic voltammetry, the electrode potential Working
Rs output
or voltage at
PS: Potentiostat
ramps linearly
versus
time pulses of constant amplitude are
Cdl

Small
 AccordingtoOutput
the ohms
datalaw
are then
plotted as current
(I) vs.
superimposed
on a linear
potential ramp
Where,
Rct=Charge
transfer
voltage (V) V = Iapplied
Z
to the working electrode
resistance
Rs= Solution resistance
Current
is sampled twice
Cdl=
layer and is a complex
Where Z is the impedance of thedouble
system
capacitor
∆I (= i2 – i1) is plotted against the applied
quantity depends on the frequency potential
of the signal
and displayed
‫׀‬Z ‫( = ׀‬ReZ)2 + (ImZ)2
30
Electrochemical behavior of Au
nanostructures
 The values of resistance for the bare Au
electrode, pyramid, spherical and rod – like
nanostructured electrodes was 20.5 KΩ, 6.5 KΩ,
2.3 KΩ and 9.8 KΩ, respectively.
 The spherical nanostructured electrode
showed the smallest value of charge-transfer
resistance

Similarly it has highest conductivity among the
three types of electrodes.
It showed the electrochemical response
strongly
depended on the surface area and
the surface coverage of the electrode.
Fig: Impedance and CV response of three
31
types of nanostructures.
Optimization of experimental
conditions
16
(a)
14
 Acidic or basic environment can affect the
Current (µA)
12
biocatalytic performance of immunosensor.
10
8
6
4
2
0
 The attachment of Carcinoembryonic
antigen (CEA) with anti CEA was done for
several hours.
3
4
5
6
8
(b)
7
Spherical
6
5
∆I (µA)
7
pH
Pyramid
4
Rod
3
2
The pH = 7.0. and 2hr time of incubation
of antibody was selected.
1
0
1
2
3
4
5
6
7
Time (hr)
Fig: Effect of (a) pH of solution and (b)
incubation time on performance
of
32
immunosensor.
Cyclic voltammetry at different steps of
immunosensor preparation

Au
Peak current of the bare 1.0x10
-5
0.0
was 8.69µA.
Pyramid
(a) Pyramid
Spherical
(b) Spherical
Rod
(c) Rod
(i) Bare Au
-5
-1.0x10
-5
1.0x10
Current (A)
0.0

The peak current
-1.0x10
increased
1.0x10
1.40 times as compared to 0.0
-1.0x10
bare Au electrode.
1.0x10
(ii) Nanostructured
-5
-5
(iii) SAM
-5
-5
0.0
(iv) aCEA
 With further modification -1.0x10
0.0
0.2
0.0
0.2
0.0
0.2
0.4
with anti CEA showed 0.83,
Voltage (V)
0.76 and 0.77 times drop in
Fig: CV response of the nanostructured immunosensor.
the peak current for pyramid,
spherical and rod like
nanostructures, respectively.
The electrode was successfully modified with cancer biomarker antibody.
-5
33
Cancer biomarker limit of
detection (LOD)
 The response of immunosensor was
investigated with different concentration of
CEA ranges from 1pg/ml to 1000ng/ml.
 The limit of detection (LOD) was
calculated using the equation
S.D = standard deviation
• Sensitivity was evaluated by the slop of current
verses concentration graph.
Nanostructures
Bare Au
Pyramid
Spherical
Rod
Sensitivity (µA ng-1.
ml)
LOD ( ng/ml)
0.211
0.339
0.457
0.312
0.07
0.0039
0.0036
0.0045
Decorating electrode with nanostructures improved the performance of
immunosensor.
34
Bode plot of nanaostructured
immunosensor
 The bode plot of different
concentration ranges from
1pg/ml to1000ng/ml.
10000
Z (Ω)
nanostructured electrode with
100000
(b) Spherical
10000
1000
1000ng/ml
100ng/ml
10ng/ml
1ng/ml
0.1ng/ml
0.01ng/ml
0.001ng/ml
Antibody
1000
1
10
100
1000
10000
1
f(Hz)
10
100
1000
10000
f(Hz)
1000ng/ml
100ng/ml
10ng/ml
1ng/ml
0.1ng/ml
0.01ng/ml
0.001ng/ml
Antibody
(c) Rod
100000
Z (Ω)
 The resistance obtained was
normalized using formula
1000ng/ml
100ng/ml
10ng/ml
1ng/ml
0.1ng/ml
0.01ng/ml
0.001ng/ml
Antibody
(a) Pyramid
Z(Ω)
100000
10000
1000
Rct(i) = Resistance of antigen
Rct(o) = Resistance of antibody
1
10
100
f(Hz)
1000
10000
Fig: Bode plot of (a) pyramid (b) Spherical (c) rod like
nanostructures.
35
Association constant
3
 Association constant tells about the binding
affinity of antibody and antigen
 The association constant was calculated
using the equation
2
RN
 It usually lie between in the range 106 to
109 M-1
Bare Au
Pyramid
Spherical
Rod
1
0
1E-3 0.01
0.1
1
10
-1
Log C (ngml )
100 1000
Fig: Normalized resistance verse concentration
Where Ka = Association constant
C = Concentration
The mean association constant for nanostructured electrode came out
to be 0.0783*109 M-1.
36
Selectivity of nanostructured
immunosensor
 The immunosensor was incubated in
solution containing HBsAg, AFP and
PSA for 2 hours of fixed concentration
of 100ng/ml.
 It can be seen from that after attachment
of cancer biomarker the current
decreased to value of 6.62µA.
Current (µA)
8
6
4
2
0
ACEA
ACEA+ CEA ACEA+ HBsAg ACEA+ AFP ACEA+ PSA
Fig: Selectivity of an immunosensor.
HBsAg: Hypatitis B virus surface antigen
AFP: α- fetoprotein
PSA: Prostate cancer antigen
The prepared immunosensor for colon cancer detection showed good
specificity.
37
Stability of nanostructured
immunosensor
12
10
14.0
8
6
4
13.0
12.5
12.0
11.5
2
0
5
10
15
20
25
30
Days
0
 The current retained 83% of the
original value even after 30 days.
13.5
Current (µA)
 Keeping electrodes at 4°C in 0.1M
phosphate buffer solution (PBS pH= 7.0)
and measurements were repeated every 3
days for a month.
14
Current (µA)
The stability of the immunosensor
decorated with spherical nanostructures
was also determined.
0
5
10
15
20
25
30
Days
Fig: Selectivity of an immunosensor.
The stability of immunosensor was also excellent.
38
Conclusions (Part I)
 Colon cancer was diagnosed using three different types of
nanostructures pyramid, spherical, and rod like nanostructures.
 The spherical nanostructures were smaller in size and has larger
value of surface area as compared to the pyramid and rod- like
nanostructures.
 Due to the higher surface area the spherical nanostructure showed
better electrochemical performance than the other types of
nanostructures.
 The prepared immunosensor for cancer detection showed LOD of 4
pg/ml and have stability almost for a month.
39
PART II
An electrochemical immunosensor for
prostate specific antigen based on polymer brush
colabeled silica nanoparticles.
Collaborators
Prof. Chang Mang Li, Dr. Hu Weihua, Dr. Wang Bin
School of Chemical and Biomedical Engineering, Nanyang Technological
University, Singapore.
40
Schematics of preparation of
sandwich immunosensor
+ Tetraethyl orthosilicate (TEOS) +
=
• THF (tetrahydrofuran)
• BIB (2-bromoisobutyryl bromide)
• TEA (triethylamine)
• OEGMA (oligo ethylene glycol
methacrylate)
• GMA (glycidyl methacrylate)
41
Optimization of experimental
conditions
90
(i) 0.5 % GMA
 The SAM was grown uniformly onto
the surface with the height of 3 nm
Current (µA)
85
 The current decreased with volume
ratio
(ii) 10 % OEGMA
(a)
80
75
70
5
10
15
OEGMA concentration (%)
(b)
0.4
0.6
GMA concentration (%)
0.8
(c)
 The height varied from3 nm to 17 nm
thus confirmed successful synthesis
of the polymer brushes.
 Prostate cancer biomarker were
immobilized on the polymer brush- AuNS electrode in the range of 450 to
1600 ngml-1
Fig: (a) Current verses volume ratio
of OEGMA-GMA (b) Surface
morphology after SAM development
(c) After polymer brush growth (d)
Peak current with concentration
of antibody
42
Morphology of Au and Silica
nanoparticles
AuNp
SiNps
SiNPs +Ab
• The diameter of the Au nanoparticles came out to be 14 ± 1 nm.
• The bare silica nanoparticles have an average size
of about 125± 2 nm.
• After modification of secondary antibody the surface become rough and no
significant change in size was observed after modification.
43
Characterization of conjugated
Silica nanoparticles
•
The differential pulse voltammetry (DPV)
measurements were performed to verify the
attachment of secondary antibody with
SiNPs.
• The current value decreased from 58 µA to
50.3 µA when SiNPs+ Ab were used.
-
• This value shift to +11 mV after modification
with TEOS.
• After conjugation of antibody this potential shifted
to -33 mV.
20
10
Zeta potential (mV)
• The value of zeta potential of bare SiNPs
25.8 mV.
Silica nanoparticles was successfully modified with
secondary antibody.
0
-10
-20
-30
Bare SiNPs
SiNPs+ GPTMS
SiNPs+ GPTMS+Ab
Fig: DPV and zeta potential measurements
44
Electrochemical measurements of
Prepared electrode
3000
1000
0
0.0
Polymer brush
2000
-Z"
Current (µA)
-5
5.0x10
2000
-Z "
-4
1.0x10
AuNS
SNS
PB
Antibody
Antigen
SiNp+ Ab
1000
-5
-5.0x10
3000
0
Antibody + SiNPs
-4
-1.0x10
-Z"
2000
-0.35 -0.30 -0.25 -0.20 -0.15 -0.10 -0.05 0.00 0.05
Potential (V)
1000
0
1000
2000
3000
4000
Z'
(a)
(b)
(c)
(a) Cyclic voltammetry (b- c) impedance response at different steps of preparation of sandwich immunosensor .
•
The value of peak current for AuPs was 93 µA.
•
The grafting of GMA-co-OEGMA polymer brush resulted decrease in current value to
75 µA.
•
Whereas current values decreased to 71, 64 and 56 µA for antibody, antigen and
SiNPs + Ab, respectively.
•
Similar results have been obtained from EIS measurements.
45
Analytical performance of
immunosensor: EIS study
3000
0.005ngml-1
0.01 ngml-1
0.03 ngml-1
0.06 ngml-1
0.1 ngml-1
-1
1ngml
-1
10ngml
-1
100ngml
1500
-Z" (Ω)
30000
1500
30000
1000ngml-1
1500
0
0
2000
4000
6000
0
1500
3000
4500
6000
1500
3000
4500
6000
7500
Z' (Ω)
•
Nyquist plot for different concentration of antigen ranges from 5 pg to 1000ng/ml.
Concentration 0.005
(ng/ml)
0.01
0.03
0.06
0.1
1
10
100
1000
Rct (Ω)
1699
2195
2616
3269
4251
5624
6200
46
7356
1156
Cont……….
Rct(2) = Resistance of antigen
Rct(1) = Resistance of antibody
Bare Au
Nanostructured electrode
7
Normalized resistance
• The resistance was normalized by using the
formula given by
6
5
4
3
2
1
• It showed that it possessed a linear
relationship with concentration
0
0.01
0.1
1
10
log (C) (ngml-1)
100
1000
The resistance increases with increase in concentration. The dynamic
range is different for bare and Ns surface.
47
Differential Pulse measurements
with
•
The sensitivity came out to be 2.3375 and 4.9333 µA
pg-1ml for bare and nanostructured
electrode.
•
The theoretical value of limit of detection was
evaluated using the equation
-5
8.0x10
Current (A)
• The peak current decreased from 96 to 55 µA
the increase in concentration.
-4
1.0x10
-5
6.0x10
-5
0.005ng/ml
0.01ng/ml
0.03ng/ml
0.06ng/ml
0.1ng/ml
1ng/ml
10ng/ml
100ng/ml
1000ng/ml
4.0x10
-5
2.0x10
0.0
-0.35 -0.30 -0.25 -0.20 -0.15 -0.10 -0.05 0.00
Voltage (V)
100
LOD= 3× S.D/ sensitivity
Bare Au
Nanostructured electrode
Where S.D = Standard deviation
Dynamic range
(pgml-1-ngml1)
LOD
(pgml-1)
Sensitivity
(µApg1ml)
Bare Au 30-1000
10
2.3
NS Au
2.3
4.9
5-1000
Current (µA)
90
80
70
60
50
1E-4
0.01
1
log (C) (ngml-1)
100
Fig: Current verse logarithm of concentration
48
Specificity of sandwich
immunosensor
• The specificity towards prostate cancer was
checked by using some other cancer
biomarkers.
•
The other cancer biomarkers used were CEA,
AFM1, IgG and AFP.
The change in current
10
∆I (µA)
•
12
8
6
4
ΔI = I2- I1
where I2 = Peak current of PSA antibody
I1 = After attachment with PSA and other
antigens.
2
0
PSA
The specificity of prostate cancer detection was quite
good.
CEA
AFM1
IgG
AFP
Fig: Specificity of immunosensor
PSA: Prostate specific antigen
CEA: Carcinoembryonic antigen
AFM1: Anti- Aflatoxin M1
IgG: Immunoglobin G
AFP: α- Feto protein
49
Conclusion (Part II)
• A prostate cancer was successfully diagnosed using polymer
brush based sandwich immunosensor.
• The prepared sandwich immunosensor was found to detect the
prostate cancer in the concentration range of 5pg/ml to 1000ng/ml .
• The sandwich immunosensor show 2.3375 and 4.9333 µA pg-1ml
sensitivity for bare Au and nanostructured electrode.
• The limit of detection came out to be 2pg/ml which is less as
compared to the bare Au electrode.
• The immunosensor so prepared showed good LOD, sensitivity
and specificity.
50
Publications
•
S. Rafique, C. Gao, C. M. Li, and A. S. Bhatti, Comparative study of label-free
electrochemical immunoassay on various gold nanostructures, J. Appl. Phys. 114, (2013) ,
164703-164713.
•
S. Rafique, W. Bin, A. S. Bhatti, Silica nanoparticles labeled polymer brush
electrochemical
Sensors &
•
immunosensor for prostate specific antigens, prepared and submitted in
Actuators B.
S. Rafique and A. S. Bhatti, Improvement in adhesion and elastic properties of
agglomerated self assembled monolayers by annealing, under process of submission.
•
A. S. Bhatti, H. Habib, S. Mehmod, S. Rafique and A. Naeem, The kinetics and force
spectroscopy of self assembled monolayer and GC contents modified DNA, under process
of submission.
51
Conference Presentations
• “Second Conference on Nanotechnology for Biological and Biomedical Applications (Nano-Bio-Med 2013),
14 – 18 October 2013, Trieste, Italy.
• “Joint International Workshop on Nanotechnology: Policy and Ethics” 25- 27 March, 2013, Islamabad,
Pakistan.
• “International Conference on Nanomaterials and Nanoethics” 01-03
Dec, 2011, Lahore, Pakistan .
• “36th International Nathiagali Summer College on Physics & Contemporary Needs” from 4th to 8th July
2011 at National Center for Physics Islamabad.
• “1st International Symposium on Nanomedicine: Past, Present & Future Prospects & Workshop on
Techniques in Nanomedicine Research” 20th to 24th December 2010, H.E.J. Research Institute of Chemistry
International Center for Chemical and Biological Sciences University of Karachi, Pakistan. (Best poster award)
•
“1st Biosciences poster competition and exhibition (BioPEC) 2010” 20th May, 2010 at COMSATS Institute of
Information Technology, Islamabad, Pakistan.
• “International workshop on Application Nanotechnology (WANT) 2010” 31st May- 4th June, 2010 at
National
Centre for Physics, Islamabad, Pakistan.
• “35th International Nathiagali Summer College on Physics and Contemporary Needs” 28 June -10 July,
2010
Nathiagali, Pakistan
• “1st BICMAP, CIIT Science Conference 2009” July 28th -29th, 2009 at COMSATS Institute of Information
52
Technology, Abbottabad, Pakistan.
Acknowledgements
Higher Education
Commission of Pakistan
COMSATS Institute of
Information Technology
NANYANG Technological
University Singapore
Center for Micro and
Nano Devices
53
Thanks
54