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