Metallomics Study of Lead-Protein Interactions in Albumen by Electrochemical and Electrophoretic
Electrochemical Studies of Nanostructured Protein Based...
Transcript of Electrochemical Studies of Nanostructured Protein Based...
Electrochemical Studies of Nanostructured Protein Based Immunosensors.
COMSATS Institute of Information Technology Islamabad, Pakistan.
BySaima Rafique
Supervised byProf. Arshad Saleem Bhatti
Center for Micro and Nano devices, Dept. of Physics, COMSATS Institute of Information Technology, Islamabad.
Collaborators
Prof. Chang Mang LiSchool of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
1
Outline Introduction Cancer Cancer biomarkers Immunosensors Importance of Supporting matrix for immunosensor
Section I(Growth kinetics and adhesion behavior of self assembledmonolayers (SAM))
Steps for preparation Surface morphology (AFM) Growth kinetics
f-d Curves Conclusions
Acknowledgements
Section II(Early stage cancer diagnostics using cancer
biomarkers)PART I (Colon cancer) 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 Conclusions
2
Motivation
How frequently people go for regular medical check up
Cancer controlPrimary prevention
• Public information and education• Self examination
Secondary prevention • Early diagnosis• Screening and therapy
In Pakistan most common cancers are
• Breast Cancer (22% of all cases) • Lung cancer (18%)• Prostate cancer (13%)• Colon (8%)
N. Bano et al, Asian J Pharm Clin Res Ϯ 6, (2013), 13-17.*A. Gul et al, J. Med. Sci. 20, (2012), 67-70.M. Hanif et al, Asian Pac. J. Cancer. Pre. ǁ 10, (2009), 227-230.
Ϯ
3
*Cancer statistics in Pakistan
22%
18%14%13%
12%8%
6%4% 3%
Cancer statistics in PakistanBreast
Lungs
Blood
Prostate
Others
Colon
LiverBrain
Bone
4
Cancer: An introduction
Disease caused by an uncontrolled growth of abnormal cells in a part of the body
*A. Mishra and M. Verma, Cancers, 2, (2010), 190-20.
*
Cancer biomarkers
• A cancer biomarker refers to a
substance that is indicative of the
presence of cancer in the body
Different types of cancer biomarkers
• 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)
5
Cancer detection: Enzyme-linked immunosorbent assay (ELISA)
The ELISA has been used as a diagnostic tool in medicine
Different types of ELISA are
• Direct assays• Indirect assays• Sandwich assays
Antibody (Ab)
Antigen (Ag)
Disadvantages
Enzyme reaction is short term so signal must be read as soon as possible.
It being an enzymatic reaction, even small quantities of non-specific binding might result in false signal.
ELISAs is quite complex, including multiple steps of incubation and washing.
Fig: ELISA
*M. Thompson, Anal. Meth., 45, (2010), 1757-1759.
*
Label free Immunosensor
Immunosensors are biosensors based on specific antigen-antibody interactions
It eliminates the need for tags
Simplifying assay designed
Fast detection
6
• Current• Impedance
Supporting Matrix Antibody Antigen
Fig: Schematics of immunosensor
*A. Sharma , Z. Matharu, G. Sumana, Thin Solid Films, 38, (2010), 245-251
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
Importance of supporting matrix for immunosensor
Fig: Various types of supporting matrix7
Self assembled monolayers Polymer brushes Gold NPs
Carbon nanotunes
Supporting matrix: Self assembled monolayer (SAM)
Fig: Schematics of 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
head group of selected organic molecule.
*Christopher. J. Love, L. A. E, Chem. Rev, 105 (2005) 1103-1169 8
Advantage of using SAM is it reduces physical adsorption of biomolecules.
Supporting matrix: Gold nanoparticles
The different nanomaterials are developed, among them
gold nanoparticles (AuNPs) has been frequently used
because of
• Easy functionalization
Good biocompatibility
Fig: Various shapes of gold nanostructures
*S. Barua, J. Yoo and S. Mitragotri, Proc. Natl. Acad. Sci., 110, 3270 (2013). 9
They provide high surface area, more no of biomolecules can be attached.
Supporting matrix: Polymer brushes
Homopolymer (a)
Copolymer
Alternating (b)(BABABABA)
Random (c)(BBBABBBABA)
Block (d)(BBBAAABBBAAA)
Graft copolymers (e) (A-A-A-A-A-A-A-A-A-A-A-A-A-A-A)
B-B-B-B B-B-B-B
Polymer brush is a layer of polymers attached with oneend to a surface.
Homo polymer brushes
10High density of polymer brushes reduces the physical adsorption.
(a) Polytetrafluoroethylene (PTFE)(b) Poly(methyl methacrylate) and poly(N-isopropylacrylamide) [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)
Aim of Study
The aim of the present research work is to
Study the growth kinetics and adhesion parameters of self assembled monolayers
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
Washed and rinsed with ethanol
Chip incubation in SAM*+ EthanolSolution (1, 3, 5, 7, 9hrs)Washed + DriedAnnealing SAM at 100°C for 1hr
Annealed at 300°C for 3 hr
• Two sets of samples were prepared one as grown and other is annealed
14
*SAM= 16- Mercapto-1-hexadecanol
AFM analysis
As grown SAM t= 1, 5 ,9hrs
Annealed SAMt= 1, 5, 9 hrs
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.
15
Conti….
10
20
30
40
50
60
(a) As - grown
15
18
21
24
0 1 2 3 4 5 6 7 8 9 10
5
10
15
20
Surf
ace
cove
rge
(%)
Th
ick
nes
s (n
m)
(b) Annealed
25
30
35
40
45
50
Incubation time (hr)Fig: Thickness and surface coverage verses incubation time
As the thickness increased the surface
coverage saturates to 22% after 3hrs of incubation.
In case of annealed SAM, the thickness
dropped significantly and doubled the surface coverage.
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.
16
Aspect ratio
0 2 4 6 8 100.0
0.5
1.0
1.5
2.0
2.5
3.0
Ave
rage
(T
hic
kn
ess/
surf
ace
cove
rage
)
Incubation Time (hrs)
As-grown
Annealed
Fig: Aspect ratio verses time
The aspect ratio is
For as grown SAM the aspect ratio
increased due to the increase in thickness with the incubation time.
For annealed SAM it reduced significantly
due to decreased in thickness as well
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
(b) The adsorbed SAM acts as a
nucleation sites so multilayer formation
increased
(c) Coalescence of molecules increased
In case of annealed SAM, as incubation
time increased the relative change in
density decreased which showed that
molecules diffused as a larger grain on
adsorption site
Fig: Growth kinetics of islands
18
For as – grown SAM, the number of islands decreased and their average size increased with the increased incubation time.
0 2 4 6 8 10
90
100
110
120
130
140
150
160
170
(c)
(b)
N= 45
N= 60N= 82N= 93
N= 74
N= 170N= 174N= 210N= 295N= 303
Ave
rage
gra
in s
ize
(nm
)
Incubation time (hour)
As grown SAM
Annealed SAM
(a)
19
Atomic force microscopy (AFM)
*M. Brogly, O. Noel, H. Awada, G. Castelein and J. Schultz, C. R. Chimie ,9 (2006), 99–110
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).
At B, it approach toward the surface, thetip interacts with the sample and a jump incontact occurs
At C, embed in the surface
At D, move away towards the surface
At E, Back to normal positionFig: Force distance spectroscopy
Fig: Atomic force microscopy
F-d curves
Fig: F-d cures by AFM
• The figure shows the f-d curves for 1, 5, 9 hrs.
• It shows the variation in
loop energy
slope of the loop
pull off/ adhesion force
The morphology of SAM effects the force distance curves.20
-50
0
50
100
-100
-50
0
50
100
For
ce (
nN
)0 100 200 300
-150
-100
-50
0
50
100
150
Distance (nm)
0 100 200 300 400-150
-100
-50
0
50
100
-100
-50
0
50
100
For
ce (
nm
)
-8
-4
0
4
8(a) 1 hour
(b) 5 hours
(c) 9 hours
Annealed
As Grown
Adhesion properties of SAM
0.0 0.5 1.0 1.5 2.0 2.5 3.00.0
0.5
1.0
1.5
2.0
2.5
3.0
Loo
p E
ner
gy (1
0-14 J
) Annealed
As - grown
Aspect ratio
0.0 0.5 1.0 1.5 2.0 2.5 3.00
20
40
60
80
100
120
(c)
(b)
Pu
ll of
f fo
rce
(nN
)
Aspect ratio
Annealed
As grown
(a)
Fig: Loop energy and pull off force verses aspect ratio
The pull off force is the force required to pull off
the tip from the surface
(a) Initially, the SAM was assembling or lying onthe surface (b) As agglomeration increased it
approached to a limiting value of 38 nN (c) Further, coalescence of molecules increased the pull off force
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.The increase in the pull off force and loop energy with increasing aspect ratio is a clear indication of the agglomeration of molecules on the surface. 21
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
Maugis analyzed both the JKR and DMT models and suggested that the ǁ
transition between these models by dimensionless parameter
*R. W. Carpick et al, J. Colloid. Inter. Sci., 211, (1999), 395–400.O. D. S. Ferreira et al, Applied Surface Science, Ϯ 257 (2010), 48-54.J. P. Aim et al, J. Appl. Phys. ǁ 76, (1994), 754-762.
22
WhereZo= equilibrium separation between tip and sampleR= Tip radiusK= Elastic modulus of tipWA= Work of adhesion
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
0.0 0.5 1.0 1.5 2.0 2.5 3.00
1
2
3
4
Ela
stic
mod
ulou
s (G
Pa)
Aspect ratio
Annealed
As - grown
Fig: Elastic modulus verses aspect ratio
The elastic modulus essentially depends
upon the particle size as well as thickness
of the layers.
For annealed SAM it reached to the value
of 3.3 GPa which give rise the step difference
of 1 GPa between as grown and annealed
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 IIPart I
Comparative study of label-free electrochemical immunoassay on various gold nanostructured
electrode
Collaborators
Prof. Chang Mang Li, Dr. Gao ChuxianSchool 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 -0.08 228
Surface morphology
Fig: FESEM and AFM images of pyramid, spherical and rod like nanostructures.
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 area (µm2)
Surface 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. 29
30
Electrochemical impedance technique
Impedance methods involves a small
amplitude sinusoidal signal to system under
investigation and measure the impedance, current
or voltage at output
According to the ohms law
V = I Z
Where Z is the impedance of the system and is a complex
quantity depends on the frequency of the signal
2 + (ImZ)2(ReZ) = ׀ Z׀
IV
PS
Control
Reference
Working
CounterPS: Potentiostat
EIS data analysis
Equivalent circuit model Most common used model is
Where, Rct=Charge transfer resistance
Rs= Solution resistanceCdl= double layer
capacitor
Rs
Rct
Cdl
Cyclic voltammetry
In cyclic voltammetry, the electrode potential ramps linearly versus time Output data are then plotted as current (I) vs. voltage (V)
Differential pulse voltammetry
Small pulses of constant amplitude are superimposed on a linear potential ramp
applied to the working electrode Current is sampled twice∆I (= i2 – i1) is plotted against the applied potential and displayed
Electrochemical behavior of Au nanostructures
Fig: Impedance and CV response of three types of 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.
31
Optimization of experimental conditions
3 4 5 6 7 80
2
4
6
8
10
12
14
16
pH
Cu
rren
t (µ
A)
(a)
1 2 3 4 5 6 70
1
2
3
4
5
6
7
Rod
Pyramid
Time (hr)∆I
(µA
)
Spherical
(b)
Fig: Effect of (a) pH of solution and (b) incubation time on performance of immunosensor.
Acidic or basic environment can affect the biocatalytic performance of immunosensor.
The attachment of Carcinoembryonic antigen (CEA) with anti CEA was done for several hours.
The pH = 7.0. and 2hr time of incubation of antibody was selected.
32
Cyclic voltammetry at different steps of immunosensor preparation
Fig: CV response of the nanostructured immunosensor.
Peak current of the bare Au
was 8.69µA.
The peak current increased
1.40 times as compared to bare Au electrode.
With further modification with anti CEA showed 0.83, 0.76 and 0.77 times drop in
the peak current for pyramid, spherical and rod like nanostructures, respectively.
The electrode was successfully modified with cancer biomarker antibody.
-1.0x10-5
0.0
1.0x10-5
(a) Pyramid (b) Spherical
(c) Rod
-1.0x10-5
0.0
1.0x10-5
Cu
rren
t (A
)
-1.0x10-5
0.0
1.0x10-5
0.0 0.2
-1.0x10-5
0.0
1.0x10-5
0.0 0.2
Voltage (V)
0.0 0.2 0.4
(iv) aCEA
(iii) SAM
(ii) Nanostructured
(i) Bare Au
33
Pyramid Spherical Rod
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)
0.211 0.339 0.457 0.312
Decorating electrode with nanostructures improved the performance of immunosensor. 34
LOD ( ng/ml) 0.07 0.0039 0.0036 0.0045
Bode plot of nanaostructured immunosensor
1 10 100 1000 10000
1000
10000
100000
Z
(Ω)
f(Hz)
1000ng/ml 100ng/ml 10ng/ml 1ng/ml 0.1ng/ml 0.01ng/ml 0.001ng/ml Antibody
(a) Pyramid
1 10 100 1000 10000
1000
10000
100000
Z(Ω
)
f(Hz)
1000ng/ml100ng/ml 10ng/ml 1ng/ml 0.1ng/ml 0.01ng/ml 0.001ng/ml Antibody
(b) Spherical
1 10 100 1000 10000
1000
10000
100000
Z (
Ω)
f(Hz)
1000ng/ml 100ng/ml 10ng/ml 1ng/ml 0.1ng/ml 0.01ng/ml 0.001ng/ml Antibody
(c) Rod
Fig: Bode plot of (a) pyramid (b) Spherical (c) rod like nanostructures.
The bode plot of different nanostructured electrode with concentration ranges from 1pg/ml to1000ng/ml.
The resistance obtained was normalized using formula
Rct(i) = Resistance of antigen Rct(o) = Resistance of antibody
35
Association constant
Association constant tells about the binding affinity of antibody and antigen
It usually lie between in the range 106 to 109 M-1
The association constant was calculated using the equation
Where Ka = Association constant C = Concentration
The mean association constant for nanostructured electrode came out to be 0.0783*109 M-1.
36
1E-3 0.01 0.1 1 10 100 10000
1
2
3
Log C (ngml-1)
RN
Bare Au Pyramid Spherical Rod
Fig: Normalized resistance verse concentration
Selectivity of nanostructured immunosensor
0
2
4
6
8
ACEA+ PSAACEA+ AFPACEA+ HBsAgACEA+ CEAACEA
Cur
rent
(µA
)
Fig: Selectivity of an 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.
The prepared immunosensor for colon cancer detection showed good specificity.
37
HBsAg: Hypatitis B virus surface antigenAFP: α- fetoproteinPSA: Prostate cancer antigen
Stability of nanostructured immunosensor
0 5 10 15 20 25 300
2
4
6
8
10
12
14
0 5 10 15 20 25 30
11.5
12.0
12.5
13.0
13.5
14.0
Cu
rren
t (µ
A)
Days
Cur
rent
(µA
)
Days
Fig: Selectivity of an immunosensor.
The stability of the immunosensor decorated with spherical nanostructures was also determined.
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.
The current retained 83% of the original value even after 30 days.
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 BinSchool of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
40
Schematics of preparation of sandwich immunosensor
41
• THF (tetrahydrofuran)• BIB (2-bromoisobutyryl bromide)• TEA (triethylamine)• OEGMA (oligo ethylene glycol methacrylate)• GMA (glycidyl methacrylate)
+ Tetraethyl orthosilicate (TEOS) + =
(c)
Optimization of experimental conditions
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
(b)
The current decreased with volume ratio
The SAM was grown uniformly onto the surface with the height of 3 nm
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- Au-NS electrode in the range of 450 to 1600 ngml-1
42
5 10 15
70
75
80
85
90
Cur
rent
(µA
)
OEGMA concentration (%)
(i) 0.5 % GMA (a)
0.4 0.6 0.8
(ii) 10 % OEGMA
GMA concentration (%)
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
-30
-20
-10
0
10
20
Zet
a p
oten
tial
(m
V)
Bare SiNPs SiNPs+ GPTMS SiNPs+ GPTMS+Ab
• 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.
• The value of zeta potential of bare SiNPs -25.8 mV.
• This value shift to +11 mV after modification with TEOS.
• After conjugation of antibody this potential shiftedto -33 mV.
Fig: DPV and zeta potential measurementsSilica nanoparticles was successfully modified with secondary antibody. 44
-0.35 -0.30 -0.25 -0.20 -0.15 -0.10 -0.05 0.00 0.05
-1.0x10-4
-5.0x10-5
0.0
5.0x10-5
1.0x10-4
SNS PB Antibody Antigen SiNp+ Ab
Cur
ren
t (µA
)
Potential (V)
0
1000
2000
3000 AuNS
-Z"
0
1000
2000
Polymer brush
-Z"
1000 2000 3000 40000
1000
2000
3000 Antibody + SiNPs
-Z"
Z'
Electrochemical measurements of Prepared electrode
(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
• Nyquist plot for different concentration of antigen ranges from 5 pg to 1000ng/ml.
Concentration(ng/ml)
0.005 0.01 0.03 0.06 0.1 1 10 100 1000
Rct (Ω) 1156 1699 2195 2616 3269 4251 5624 6200 7356
0
1500
3000
0.005ngml-1
-Z"
(Ω)
0.01 ngml-1
0.03 ngml-1
0
1500
3000
0.06 ngml-1 0.1 ngml-1 1ngml-1
0 2000 4000 60000
1500
3000
10ngml-1
Z' (Ω)
0 1500 3000 4500 6000
100ngml-1
1500 3000 4500 6000 7500
1000ngml-1
46
Cont……….
0.01 0.1 1 10 100 10000
1
2
3
4
5
6
7
log (C) (ngml-1)
Nor
mal
ized
res
ista
nce
Bare Au Nanostructured electrode• The resistance was normalized by using the
formula given by
Rct(2) = Resistance of antigenRct(1) = Resistance of antibody
• It showed that it possessed a linear relationship with concentration
The resistance increases with increase in concentration. The dynamic range is different for bare and Ns surface.
47
Differential Pulse measurements
-0.35 -0.30 -0.25 -0.20 -0.15 -0.10 -0.05 0.000.0
2.0x10-5
4.0x10-5
6.0x10-5
8.0x10-5
1.0x10-4 0.005ng/ml 0.01ng/ml 0.03ng/ml 0.06ng/ml 0.1ng/ml 1ng/ml 10ng/ml 100ng/ml 1000ng/ml
Voltage (V)
Cur
rent
(A
)1E-4 0.01 1 100
50
60
70
80
90
100 Bare Au Nanostructured electrode
log (C) (ngml-1)C
urre
nt
(µA
)
• The peak current decreased from 96 to 55 µA with the increase in concentration.
• 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
LOD= 3× S.D/ sensitivity
Where S.D = Standard deviation
Fig: Current verse logarithm of concentration48
Dynamic range (pgml-1-ngml-1)
LOD(pgml-1)
Sensitivity(µApg-1ml)
Bare Au 30-1000 10 2.3
NS Au 5-1000 2.3 4.9
Specificity of sandwich immunosensor
0
2
4
6
8
10
12
∆I (µ
A)
AFPIgGAFM1CEAPSA
• 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
ΔI = I2- I1
where I2 = Peak current of PSA antibody I1 = After attachment with PSA and other antigens. Fig: Specificity of immunosensor
The specificity of prostate cancer detection was quite good.
49
PSA: Prostate specific antigenCEA: Carcinoembryonic antigenAFM1: Anti- Aflatoxin M1IgG: Immunoglobin GAFP: α- Feto protein
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 immunosensor for prostate specific antigens, prepared and submitted in
Sensors & 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
52
• “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 Technology, Abbottabad, Pakistan.
Acknowledgements
COMSATS Institute of Information Technology
Center for Micro and Nano Devices
Higher Education Commission of Pakistan
NANYANG Technological University Singapore
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Thanks