1. Reporter : Hsun-Pei Wu Advisor : Cheng-Hsin Chuang Department of Mechanical Engineering & Institute of Nanotechnology, Southern Taiwan University, Tainan, TAIWAN March 28th Enhancement of Fluorescent Intensity by Using DEP Manipulations of Polyaniline-coated Al 2 O 3 Nanoparticles for Immunosensing

2. Outline 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 -Al 2 O 3 NPs  Synthesis of PANDB- Al 2 O 3 NPs  TEM Image and FTIR Speatr of NPs

3. Motivation Dot-Electrode Array Genomics Proteomics Protein analysis Two-Dimensional Gel Electrophoresis, 2-DE Multi-dimensional Liquid Chromatography, MDLC Mass Spectrometry Disadvantage  Expensive  Consuming Protein array chips Western blot Nitrocellulose Magnetic Beads Disadvantage  External Magnetic Field  Chip Size limit Dielectrophoretic (DEP) Biochip Disadvantage  Expensive  Consuming Advantage Rapidly High-Sensitivity Control the Single-Point Not limited to particle types Polyaniline-coated Al 2 O 3 Nanoparticles PANDB Layer Al 2 O 3

4. Positive DEP Force GND 10V ITO Glass Glass Au With/Without DEP Force BSA Flow Direction Flow O ∥ O ∥ CH - CH 2 - CH 2 - CH 2 - CH - CH - NH 2 PANDB Layer -NH 2 PANDB-Al 2 O 3 -NPs Al 2 O 3 Silane-Al 2 O 3 -NPs -NH 2 Glutaraldehyde Antibody Al 2 O 3 Fluorescent protein Sensing Mechanism ImmunosensingImmunosensing Fluorescent Intensity

5. 5 Negative dielectrophoresis Positive dielectrophoresis +V -V Neutral particle Net Force DEP Theory Dielectrophoresis- Motion of a particle produced by the interaction of a Non-uniform electric field with the induced effective dipole moment of the particle. Clausius-Mossotti factor Re(f CM )>0 ; Positive DEP Re(f CM )<0 ; Negative DEP

6.  Four Different forms of PANI Reduced State Oxidized State Redox State Forms Pernigraniline Base (PNB) Metallic Emeraldine Salt (ES) Emeraldine Base (EB) Leuco emeraldine Base (LEB) Chemical Formula Dodecyl Benzene Sulfonic Acid (DBSA) HClAcOHH 3 PO 4 DBSA 10 4 S/cm10 1 S/cm Doping Different Acid Without corrosion of metal electrodes by the residual acid Good Adhesion Better soluble for synthesis Without corrosion of metal electrodes by the residual acid Good Adhesion Better soluble for synthesis Scheme of the molecular formula of PANDB TEM image of PANDB  Different Forms  Different Electrical Properties

7.  Synthesis of Al 2 O 3 NPs

8. TEM Image of PANDB-coated Al 2 O 3 NPs Silane-Al 2 O 3 -NPs PANDB-Al 2 O 3 -NPs Al 2 O 3 Silane Al 2 O 3 PANDB Layer Size: 10-20 nmSize: 13-25 nm

9. FTIR spectra of Silane Al 2 O 3 and PANDB Funtional groupWavenumber(cm -1 ) -NH 2 3444 -CH32958 -CH2-2920 -C=C1467 Silane-Al 2 O 3 -NPs PANDB-Al 2 O 3 -NPs Al 2 O 3 Silane Al 2 O 3 PANDB Layer Funtional groupWavenumber(cm -1 ) -Si-O-Al 810 、 1076 -Si-OH920 -NH 2 1560 Al-OH3400 1550 820 3480 (a) (b) (c) FTIR of (a) Silane; (b) Pure-Al 2 O 3 ; (c) Silane-Al 2 O 3 -NH 2

10. Fabrication of DEP Chip 56 789 4 123 DEP Chip

11. Experimental Setup GND 56 789 4 123 DEP Chip Outlet Inlet Function Generator Voltage: 10 Vpp Frequency: 10 ~ 50 kHz CCD Camera Fluorescent Image Fluorescent Intensity Syringe Pumps LabVIEW Program 56 789 10 123 4 Circuit Board DAQ System

12. Experimental Result- Experimental Result- (1) Fluorescent intensity Fluorescent images of the difference between with DEP force and without DEP force in immunosensing Silane-Al 2 O 3 -Antibody-BSAPANDB-Al 2 O 3 -Antibody-BSA Immunosensing with DEP Immunosensing without DEP Immunosensing with DEP Immunosensing without DEP DEP Chip of OM Image SEM Image of Al 2 O 3 NPs SEM Image of PANDB-Al 2 O 3 NPs

13. 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-Al 2 O 3 -NPs are trapping with /without DEP PANDB-Al 2 O 3 -NPs are trapping with /without DEP 1 Times Without DEP With DEP 4 Times Saturated after 20 min Experimental Result- Experimental Result- (2) Effect of Immunosensing Time

14. Conclusions  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.

15. chchuang@mail.stut.edu.tw