A NOVEL LABEL-FREE ELECTROCHEMICAL IMMUNOSENSOR FOR CARCINOEMBRYONIC ANTIGEN DETECTION BASED ON THE [AGAG2O]/SIO2 NANOCOMPOSITE MATERIAL AS A REDOX PROBE

Introduction

Biosensor

Methods

Materials

BIOSENSOR

Electroactive substance

Electric signal

PH change Heat Light Mass change

Signal transducers Immobilization layer

CARCINOEMBRYONIC ANTIGEN
# Glycoprotein involved in cell adhesion # Normally produced during fetal development, but the production of CEA stops before birth. Higher levels Individuals with carcinoma tumor (colorectal , gastric, pancreatic, lung, breast, and thyroid) had higher levels of CEA than healthy individuals. Regions of high CEA levels in the body can be detected with the anti-CEA. However CEA levels may also be raised in some nonneoplastic conditions like ulcerative colitis, pancreatitis, cirrhosis, COPD, Crohn's disease as well as in smokers.
http://journals.cambridge.org/fulltext_content/ERM/ERM2_03/S146239940000168Xsup016.gif

METHODS

Three Electrode System

Reference electrode Auxiliary electrode Working electrode

http://en.wikipedia.org/wiki/Voltammetry

Cyclic Voltammetery
Oxidation peak

Finish Start

Reduction peak
http://www.als-japan.com/xdata/electorode_photo/CV_figure_1.jpg
http://www.cheng.cam.ac.uk/research/groups/electrochem/JAVA/electroche mistry/ELEC/l4fig/fig6.gif

Transmission Electron Micrograph (TEM)

# Using a beam of electron. # Capable of imaging at a significantly higher resolution than light microscopes. # Examine fine detail as small as single column of atoms. # 2-D photo.
http://upload.wikimedia.org/wikipedia/commons/6/61/Scheme_TEM.gif

Scanning Electron Microscope (SEM)

# Using a beam of electron. # Images the sample surface. # Producing signals that contain information about the sample surface topography, composition.. # 3-D photo.
http://www.purdue.edu/rem/rs/graphics/sem2.gif

IMMUNOSENSOR

= Glassy Carbon Electrodes = Nanocomposite

Y Y

= Bovine Serum Albumin = Nano Au = Anti CEA = BSA

The fabrication of the immonusensors

Nanocomp osite

BSA

Nano Au

Y
Y Anti CEA Y

Y Y Y Y BSA Y Y Y Y

Y Y Y Y Y Y

Glassy Carbon Electrode (GCE)

The working electrode in this experiment

Properties:
# High temperature resistance # Hardness # Low density # Low electrical resistance # Low friction # Low thermal resistance # Extreme resistance to chemical attack # Impermeability to gases and liquids
http://www.gpinstruments.com/shop/products_pictures/tn_GCE_on_White.jpg

The fabrication of the immonusensors

Nanocomposite

Nanocomposite

NANOCOMPOSITE
# Solid material with # Size: less then 100 nm

= nano-SiO2 = APTMS = nano-Ag = Ag-Ag2O

Nanocomposite

What are the nano particles?

Nano SiO2:
Ammonia aqueous solution (28%) Absolute ethanol Clear solution

Clear solution tetraethylorthosilicate (TEOS)

Nano SiO2

Opaque whit solution

Why to use SiO2?

* Excellent biocompatibility. * Strong adsorption ability. * Possesses abundant OH groups. ~60 nm
J. Zou, Y. Xu, B. Hou, D. Wu, Y.H. Sun, Powder Technol. 183 (2008)

Nanocomposite

3-Aminopropyltrimethoxysilane (AMPTS)

Nanocomposite

The preparation of the [Ag-Ag2O]/SiO2 nanocomposite material

APTMS Nano-SiO2

Danit Atias a,b,Yael Liebesc,Vered Chalifa-Caspid, Laetitia Bremande, Leslie Lobel a, Robert S. Marksb,d,f, , Phillipe Dussart

Nanocomposite

The preparation of the [Ag-Ag2O]/SiO2 nanocomposite material

APTMS Nano-SiO2

AgNO3

AgNO3

Ag+

NO3

J. Zou, Y. Xu, B. Hou, D. Wu, Y.H. Sun, Powder Technol. 183 (2008) 122-126

Nanocomposite

The preparation of the [Ag-Ag2O]/SiO2 nanocomposite material

APTMS Nano-SiO2

AgNO3

Nano Ag seed

Nanocomposite

Nano Ag:
Furnace Quartz tube reactor polished silicon wafer 600oc 600oC Ag/Chitosan 10 -150 nm

Air

Nitrogen

G. Frens, Nat. Phys. 241 (1973) 2024

Nanocomposite

The preparation of the [Ag-Ag2O]/SiO2 nanocomposite material

APTMS Nano-SiO2

AgNO3

Nano Ag seed

NaBH4

4Ag+ 4Ag+

BH4

4OH 4OH

4Ag

BH2(OH)2
4Ag

H2O 4H2O

BH2(OH)2

BO2

Nanocomposite

Ag-Ag2O:
Ag2O Ag 200 < T < 360oc T < 360oc Ag nanoparticle In air Ag - Ag2O nanoparticle

200 < T < 360oc

Ag

O2
T < 360oc

Ag2O

Nanocomposite

The preparation of the [Ag-Ag2O]/SiO2 nanocomposite material

APTMS Nano-SiO2

AgNO3

SiO2 Ag-Ag2O Nano Ag seed NaBH4

The fabrication of the immonusensors

Nanocomposite

The fabrication of the immonusensors

Nanocomposi te

BSA

Nanocomposite

BSA

Bovine Serum Albumin (BSA) Serum albumin protein that has numerous biochemical applications mainly:
* Provides stability
Contains: 60 Amino acid - Lysine 26 Amino acid Arginine 17 disulfide bonds - cysteine

* Providing bioactive functionalities

The fabrication of the immonusensors

Nanocomposite

BSA

The fabrication of the immonusensors

Nanocomp osite

BSA

Nano Au

Nanocomposite

BSA

Nano Au

Nano Au:

HAuCl4

Na3-citrat

Nano Au

Size chosen = 16 um

L.Y. Zhang, R. Yuan, Y.Q. Chai, S.H. Chen, N. .236231 (2006)Wang, Q. Zhu, Biochem. Eng. 28

http://www.ionicliquid.org/en/aboutus/nano.jpg

The fabrication of the immonusensors

Nanocomp osite

BSA

Nano Au

The fabrication of the immonusensors

Nanocomp osite

BSA

Nano Au

Y
Y Anti CEA Y

Y Y Y Y BSA Y Y Y Y

Y Y Y Y Y Y

Y
Nanocomposite

BSA

Nano Au

Anti CEA

Y YYY Y Y Y Y

BS A

Y YYY Y Y Y Y

Anti CEA:

Y Y

* CV scan: -0.6 0.6 V at 100 mV/s. * Working buffer Hac-NaAc *Room temperature

Oxidation peak Finish Start

Y Y

Reduction peak
http://www.cheng.cam.ac.uk/research/groups/electroc hem/JAVA/electrochemistry/ELEC/l4fig/fig6.gif

RESULTS:
Optimization conditions for immunosensor:
Temperature: Incubation time: pH :

Best temp.: 25OC

Best time: 7min

Best pH: 5.5

The electrochemical characterization of the stepwise modified electrodes: [Ag-Ag2O]/SiO2 +

BSA + nano Au [Ag-Ag2O]/SiO2 nanocomposite + BSA

Bare GCE

Two points could be summed up for utilizing nano-Au: # High volume-surface ratio allowed maximal utilization of their bioactive sites. # nano-Au could effectively promote electronic transfer.

The electrochemical characterization of the stepwise modified electrodes: [Ag-Ag2O]/SiO2 +

BSA + nano Au [Ag-Ag2O]/SiO2 nanocomposite + BSA

Bare GCE

[Ag-Ag2O]/SiO2 nanocomposite + BSA+ nano Au + anti-CEA [Ag-Ag2O]/SiO2 nanocomposite + BSA + nano Au + anti-CEA + BSA

Performance of immunosensore:
# CV response and calibration curves:
Anodic peak Linear regression Calibration plot

With increase of CEA concentration, there is a gradual reduce in peak currents

Comparsion between the developed immunosensor with known immunosensor:

? Range of linearity

The range was greatly improved

? Limit of detection

Exhibited a lower detection limit

? Redox probe

the [AgAg2O]/SiO2 redox probe largely improved the sensitivity of the electrode.

# Selectivity:
* The effect of potential interferents on the immunosensor * Rang of experiment 5 20 ng/ml.

the selectivity of the as-prepared immunosensor was acceptable

Example for 5 ng/ml:

# Stability and repeatability:

Stability Reproducibility

90 continuous measurements

10 identical experiment using 10 equally preperd electrodes.

The electrode lost only 5% of its initial response

The electrode lost only 5% of its initial response

The stability was accepted

An acceptable

# Application:
Detecting the recoveries of six CEA sample in human serum.

Examined samples by the proposed electrochemical immunosensor and the ELISA method.

CONCLUSIONS:
the proposed immunosensor exhibited: * low detection limit * wide linear range * good reproducibility * satisfactory stability

REFERENSES:
# Articals:
1. Danit Atias a,b, Yael Liebesc, Vered Chalifa-Caspid, Laetitia Bremande, Leslie Lobel a, Robert S. Marksb,d,f, , Phillipe Dussart 2. J. Zou,Y. Xu, B. Hou, D. Wu,Y.H. Sun, Powder Technol. 183 (2008) 3. G. Frens, Nat. Phys. 241 (1973) 2024 4. C.L. Hong, R. Yuan,Y.Q. Chai, Y. Zhuo, Electroanalysis 20 (2008) 21852191 5. L.Y. Zhang, R. Yuan,Y.Q. Chai, S.H. Chen, N. Wang, Q. Zhu, Biochem. Eng. 2 6.Y.K. Twu, Y.W. Chen, C.M. Shih, Powder Technol. 185 (2008) 251257. 7. G. Jalal, R.F. Fu, J.B. Allen, Nano Lett. 5 (2007) 14061412 8. L. Suchera, T. Panote, A. Punnee, W. Booncharoen, L. Chusak, K. Proespichaya, Biosens. Bioelectron. 24 (2008) 7886. 9.Y.J. Ding, J. Liu, H. Wang, G.L. Shen, R.Q. Yu, Biomaterials 28 (2007) 2147 10. S.H. Chen, R.Yuan,Y.Q. Chai, L. Xu, N. Wang, X.N. Li, L.Y. Zhang, Electroanalysis 5 (2008) 11. K.J. Feng, C.H. Sun,Y. Kang, J.W. Chen, J.H. Jiang, G.L. Shen, R.Q. Yu, Electrochem. Commun. 10 (2008) 531535. 12. H. Tang, J.H. Chena, L.H. Nie, Y.F. Kuang, S.Z. Yao, Biosens. Bioelectron. 22 . 1067 1061 (2007)

# Web:
http://journals.cambridge.org/fulltext_content/ERM/ERM2_03/S146239940000168Xsup016.gif http://journals.cambridge.org/fulltext_content/ERM/ERM2_03/S146239940000168Xsup016.gif http://www.als-japan.com/xdata/electorode_photo/CV_figure_1.jpg http://www.cheng.cam.ac.uk/research/groups/electrochem/JAVA/electrochemistry/ELEC/l4fig/fig6.gif http://upload.wikimedia.org/wikipedia/commons/6/61/Scheme_TEM.gif http://www.purdue.edu/rem/rs/graphics/sem2.gif http://www.gpinstruments.com/shop/products_pictures/tn_GCE_on_White.jpg http://www.ionicliquid.org/en/aboutus/nano.jpg

THANK YOU!