Emre Özkumur 06/19/2006

1. Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel addresed DNA microaraysFixe et al., Biosensors and Bioelectronics 19 (2004) 1591Fixe et al., App. Phys. Let. 83 (2003) 1465Fixe et al., Nanotechnology 16 (2005) 2061 Emre Özkumur 06/19/2006

2. Outline • Background: ssDNA, dsDNA, immobilization, hybridization • Difficulties with immobilization and hybridization • time issues • Electric field assisted reactions – Nanogen’s NanoChip • Effects of pulsed E-field • Production of samples • Experiments & Data • Discussion - Reasons of observed data • Elongation of DNA with ac E-field • Accumulation of DNA on chip surface

3. oxide surface Background dsDNA ssDNA hybridization immobilization

4. Immobilization

5. Immobilization 2

6. Immobilization 3

7. Hybridization

8. Hybridization 2

9. Hybridization 3

10. NanoChip by Nanogen +++++ • DC electric field applied for 5 min. • DNA is driven electrophoretically to selected pixels (400 pixels) • High concentration of DNA on the pixel increase the probability of correct binding to surface and hybridization – binding is still in random orientation

11. Pulsed Electric field • Flexible polymide substrate • 300nm thick, 2mm wide Al lines deposited with 1mm gap in between • 200nm of Al2O3 and 200nm of SiO2 are deposited • surface functionalized with sulfo-EMCS bound to NH2 groups • 5’ end of DNA has SH- (thiol) groups

12. Experiments & Data • Passive conditions: No E-Field used. Sample held in the DNA solution for 3 hours for immobilization, 12 hours for hybridization experiments. Rinsed with DI water, fluorescence intensity measured. • Active conditions: E-Field applied. 4.5ns rise time, 4.5ns fall time. • Plots are normalized to maximum of passive immobilization/hybridization • 109 improvement in both Hybridization and immobilization times was measured

13. Experiments & Data 2 • Control experiments: • Immobilization: ssDNAs has thiol (SH) groups attached to them that let’s them bind to the functionalized surface. DNA segments without these binding bridges are the negatives for the experiment • Hybridization: ssDNAs with non-complementary sequences are used as negatives of the experiment.

14. Experiments & Data 3

15. Discussion • The effects seen cannot be simply due to electrophoretic motion of charged DNA • effects are seen both for positive & negative electrodes • The DNA solution is not homogeneous; there is a significant concentration of ssDNA on surface before the pulse is applied • surface is hydrophobic; it will prefer DNA molecules more than H20 molecules • sulfo-EMCS has a small positive surface charge which attracts negatively charged DNA

16. - - - - - +++++ Discussion 2 • Application of E-field pulse reorients the ssDNA, “probably” by desorbing the molecules • it is known that application of an ac E-field in the 10-100 MHz regime causes strong polarization effects on the DNA and its surrounding. • Fourier components of the E-field pulse near 100 MHz • rapidly changing E-field may shake the charged DNA and desorb and reorient the ssDNA • 1000 base long DNA can be elongated with ac E-field in the 100-200 kHz regime. Shorter DNAs (19mers) can have a faster response • Binding is less between the interlines • elongation perpendicular to surface may have an effect • Binding with the positive voltage is higher • Negative charge of DNA is also effective

17. +++++ - - - - - - - - - - +++++ Discussion 3 • - the dielectric response time, td, is the time required for the DNA solution to completely screen the E-field = to reach the steady-state. • td = 16ns for immobilization solution • td = 20us for hybridization solution • Voltage must be applied for 10-100us • assuming the each of the electrodes and electrolyte solution forms 2 capacitors connected in series, with dielectric spacers Al2O3 and SiO2, this is in the order of time required to charge the capacitors

18. Thanks..