Reporter : Hsun-Pei Wu

Advisor : Cheng-Hsin Chuang

Department of Mechanical Engineering & Institute of Nanotechnology,

Southern Taiwan University, Tainan, TAIWANMarch 28th

Enhancement of Fluorescent Intensity by Using DEP Manipulations of Polyaniline-coated Al2O3

Nanoparticles for Immunosensing

OutlineOutline• Motivation

• Dielectrophresis (DEP)Theory

• Characteristics of Polyaniline

• Materials

• Fabrication of DEP Chip

• Experimental Setup

• Experimental Result

• Conclusions

Conductive Polyaniline (PANI) PANI-DBSA (PANDB)

Synthesis of Silane -Al2O3 NPs Synthesis of PANDB- Al2O3 NPs TEM Image and FTIR Speatr of NPs

MotivationMotivation

Dot-Electrode Array

Genomics

Proteomics

Protein analysisProtein analysis

Two-Dimensional Gel Electrophoresis, 2-DE

Multi-dimensional Liquid Chromatography, MDLC

Mass Spectrometry

Disadvantage ExpensiveConsumingProtein array chipsProtein array chips

Western blot

Nitrocellulose

Magnetic Beads

Disadvantage External Magnetic FieldChip Size limit

Dielectrophoretic (DEP)Dielectrophoretic (DEP)

Biochip

Disadvantage ExpensiveConsuming

Advantage Rapidly High-Sensitivity Control the Single-Point Not limited to particle types

Polyaniline-coated Al2O3 Nanoparticles

PANDB Layer

Al2O3

Positive DEP Force

GND

10V

ITO Glass

GlassAu

With/Without DEP Force

BSA Flow DirectionFlow

O

∥

O

∥

CH - CH2 - CH2 - CH2 - CH - CH - NH2

PANDB Layer

-NH2

PANDB-Al2O3-NPs

Al2O3

Silane-Al2O3-NPs

-NH2

GlutaraldehydeGlutaraldehyde AntibodyAntibody

Al2O3

Fluorescent proteinFluorescent protein

Sensing MechanismSensing Mechanism

ImmunosensingImmunosensing

5

Negative dielectrophoresis Positive dielectrophoresis

+V

-V

Neutral particle

Net ForceNet Force

DEPDEP TheoryTheory

Dielectrophoresis- Motion of a particle produced by the interaction of a Non-uniform electric field with the induced effective dipole moment of the particle.

23)*m

2ε*P

ε

*m

ε*p

εRe(2 ErF mDEP

Clausius-Mossotti factor

Re(fCM)>0 ; Positive DEP

Re(fCM)<0 ; Negative DEP

Four Different forms of PANIFour Different forms of PANI

Reduced State

Oxidized State

Redox StateRedox State FormsForms

Pernigraniline Base(PNB)

Metallic Emeraldine Salt (ES)

Emeraldine Base (EB)

Leuco emeraldine Base (LEB)

Chemical FormulaChemical Formula

Dodecyl Benzene Sulfonic Acid (DBSA)

HClAcOH H3PO4DBSA

104 S/cm101 S/cm

Doping Different Acid

Doping Different Acid

Without corrosion of metal electrodes by the residual acidGood AdhesionBetter soluble for synthesis

Without corrosion of metal electrodes by the residual acidGood AdhesionBetter soluble for synthesis

Scheme of the molecular formula of PANDB

TEM image of PANDB

Different Forms Different Electrical Properties

Synthesis of AlSynthesis of Al22OO33 NPs NPs

TEM Image of PANDB-coated AlTEM Image of PANDB-coated Al22OO33 NPs NPs

Silane-Al2O3-NPsSilane-Al2O3-NPs PANDB-Al2O3-NPsPANDB-Al2O3-NPsAl2O3

Silane

Al2O3

PANDB Layer

Size: 10-20 nm Size: 13-25 nm

FTIR spectra of Silane AlFTIR spectra of Silane Al22OO33 and PANDB and PANDB

Funtional group Wavenumber(cm-1)

-NH2 3444

-CH3 2958

-CH2- 2920

-C=C 1467

Silane-Al2O3-NPsSilane-Al2O3-NPs PANDB-Al2O3-NPsPANDB-Al2O3-NPsAl2O3

Silane

Al2O3

PANDB Layer

Funtional group Wavenumber(cm-1)

-Si-O-Al 810 、 1076

-Si-OH 920

-NH2 1560

Al-OH 3400

1550

820

3480

(a)

(b)

(c)

FTIR of (a) Silane; (b) Pure-Al2O3; (c) Silane-Al2O3

-NH2

-NH2

Fabrication of DEP ChipFabrication of DEP Chip

a. Bottom Electrode Layer

Evaporate Impedance Electrode Layer

Coat PR S1813 Layer

Expose and Develop

Etching and Remove PR

b. Top Electrode Layer and Chip Bonding

Drill on the ITO Glass

Bonding

Function Generator 1

Ch 1 Ch 2GND

Ch 1: DEP Trap Nano-particle

Optical Clean Adhesive(OCA) PR S1813

Photomask

Nano-particle

Glass Bottom Electrode

ITO Glass

5 6

7 8 9

4

1 2 3DEP Chip

Experimental Setup Experimental Setup

GND

5 6

7 8 9

4

1 2 3DEP Chip

OutletInlet

Function Generator

Voltage: 10 VppFrequency: 10~50 kHz

0 10 20 30

Im m unosensing Tim e (m in)

0.8

1.2

1.6

2

2.4

No

rmal

ized

Flu

ore

scen

t In

ten

sity

( I

t /

I t =

0 )

F luorescent In tensity For Differen t Vo ltage 0 Vpp

2 Vpp

4 Vpp

6 Vpp

8 Vpp

10 Vpp

CCD Camera Fluorescent Image

Fluorescent Intensity

Syringe Pumps

LabVIEW Program

5 6

7 8 9

10

1 2 3

4

Circuit Board DAQ System

Experimental Result-Experimental Result- (1) Fluorescent intensity

Fluorescent images of the difference between with DEP force and without DEP force in immunosensing

Fluorescent images of the difference between with DEP force and without DEP force in immunosensing

Silane-Al2O3-Antibody-BSA PANDB-Al2O3-Antibody-BSA

Immunosensing with DEP

Immunosensing without DEP

Immunosensing with DEP

Immunosensing without DEP

DEP Chip of OM Image

SEM Image of Al2O3 NPs SEM Image of PANDB-Al2O3 NPs

With DEP

Without DEP

Normalized fluorescent intensity (It/It=0) for different immunosensing methods within 60 minutes, Solid lines represent the immunosensing with DEP force and dashed lines are without DEP as immunosensing

Normalized fluorescent intensity (It/It=0) for different immunosensing methods within 60 minutes, Solid lines represent the immunosensing with DEP force and dashed lines are without DEP as immunosensing

Silane-Al2O3-NPs are trapping with /without DEP

Silane-Al2O3-NPs are trapping with /without DEP

PANDB-Al2O3-NPs are trapping with /without DEP

PANDB-Al2O3-NPs are trapping with /without DEP

1 Times

Without DEP

With DEP

4 Times

0 10 20 30 40 50 60

Im m unosensing Tim e (m in)

0

2

4

6

8

No

rmal

ize

d F

luo

res

cen

t In

ten

sity

( I

t /

I t =

0 )

Comparison between Difference Methods Silane - Al2O 3 - Antibody - BSA ( without DEP )

Silane - Al2O 3 - Antibody - BSA ( with DEP )

[ PANDB-Al2O 3 ] - Antibody - BSA ( without DEP )

[ PANDB-Al2O 3 ] - Antibody - BSA ( with DEP )

Saturated after 20 min

Experimental Result-Experimental Result- (2) Effect of Immunosensing Time

ConclusionsConclusions We developed a DEP chip with a dot-electrode array for the condensation

of NPs and the enhancement of fluorescent intensity under a programmable control system.

According to the experimental results of fluorescent intensities, the intensity increased with either of applied time and immunosensing with p-DEP force.

This technology could apply to the biosensor such as immunoassay when the immobilized NPs as specific biomarkers.

In the future, our biosensor can integrate with PANDB material and impedance detection on to the immunoassay.

chchuang@mail.stut.edu.tw