1. Label Free Biomolecular Detection using Ellipsometric principles: Two Methods Jeremy Colson

2. Articles 1. Label-free detection of microarrays of biomolecules by oblique-incidence reflectivity difference microscopy - J.P.Landry, X.D.Zhu, J.P.Gregg. Optics Letters 29 6, p581 (2004) 2. Reflective interferometric detection of label-free oligonucleotides - J.Lu et al. Analytical Chem. 76, p4416 (2004) Text H.G.Tompkins and W.A.McGahan, Spectroscopic Ellipsometry and Reflectometry. John Wiley and Sons, Inc., New York, 1999.

3. Outline 1. Reflection coefficients. Ellipsometric ratio 2. Method 1: OI-RD 1. Calculations 2. Setup 3. Results 3. Method 2: RIDO 1. Setup 2. Theory 3. Results 4. Conclusion

4. Reflection Coefficients and the Ellipsometric Ratio

5. Total Reflection Coefficient for a Film on a Substrate: Phase change for one trip through film: Adding partial waves converging term:

6. OI-RD Calculations For It has been shown that * A.Wong and X.D.Zhu. Appl. Phys. A 63, 1 (1996)

7. Calculation Verification 1

8. Calculation Verification 2

9. Calculation Verification 3

10. IO-RD Calcs: What do they mean? There is a relationship between the ellipsometric phase shift from bare substrate to thin film and the quantity p - s. For dielectric constants that are real, p - s is entirely imaginary Knowing p - s and the dielectric constants, one can find d OI-RD directly measures Im( p - s )

11. OI-RD Experimental Setup 1. p-polarized He-Ne laser (632nm) 2. Photoelastic modulator oscillates polarization (50kHz) 3. Pockels cell to adjust phase difference 4. Lens focuses beam (3µm) 5. Reflection (45°) and recollimation 6. Rotatable analyzer converts oscillating polarization to oscillating intensity 7. Photodiode detects I(t)

12. OI-RD Data Collection/Calibration Procedure First and second harmonics analyzed with lock-in amplifiers Reflection off bare substrate: I(2) =0 with analyzer I() = 0 with Pockels Cell Subsequent scans: I() = phase shift ~ Im( p - s ) I(2) = Re( p - s )

13. Slide Preparation Poly-L-lysine coated glass Contact printing: 60-base oligonucleotides dissolved in water UV radiation to induce covalent bonds Washed by immersion in sodium borate buffer Hybridized in probe-mixture at 25°C for 2 h

14. Qualitative Results (a) Each column 42+µM concentration of unique DNA sequence (b) exposed to unlabeled oligonucleotides complementary to 1, Cy5- labeled oligo. complimentary to 3 (c) Cy5-fluorescence image after hybridization (d) Fig (b) - fig(a). Result: Selective binding occurs.

15. Quantitative Results Open circles: before hybridization Closed circles: after hybridization Error bars: standard deviations for four samples Leveling off => stably bound monolayer with density near saturation Im( p - s ) = 2x10 -3 => d = 1.2nm Increase of Im( p - s ) by 1.0x10 -3 => 0.6nm change in thickness

16. Reflection interferometric detection

17. RIDO Experimental Setup 1. S: 450-W Xe lamp monochromatized to ~1 nm bandwidth 2. P: s-polarizer 3. A: ~5 mm apertures (enforces collimation) 4. Incident light at 70.6° 5. D: CCD detector (Roeper Scientific)

18. Theory Setting reflection for s-polarized light to zero yields conditions for reflectivity minimum

19. Theory contd For air/SiO2/Si with 660 nm wavelength: n 1 = 1; n 2 = 1.4563; n 3 = 3.8251 For ideal conditions (perfectly flat surface, collimated monochromatic light) reflectivity changes by > factor of 10 for.22 nm thickness change at min. wavelength

20. Slide Preparation Silicon substrate with thermal oxide layer readily obtained, flat, established biomolecular attachment chemistry photoresist micropipetted onto eight spots formed 1 mm diameter dots monolayer of hydrophobic OTS applied Photoresist removed result: eight wells of bare oxide Streptavidin placed in wells, biotin-modified oligonucleotides attached to strep. layer Hybridization:target solutions pipetted into wells.

21. Preliminary data (a) patterned substrate surface with wells ~2.5 nm deep. (b) reflectivity curves for two sections after wavelength stepping (c) cross-section of wells 5-8 Calculated height of ~2.3nm matches literature values for OTS monolayer

22. Qualitative Results Outer wells did not have attachment chemistry Well 2 exposed to incorrect target Well 3 exposed to complimentary oligonucleotide sequence Wells 6 and 7 exposed to same target. Expected to bind only with 6.

23. Quantitative Results Wells 6 and 7 exposed to same target Well 6: d ~ 1.4+/-0.2 nm Well7: d ~ 0.1+/-0.2 nm Integration of topology yields total DNA in well

24. Conclusion measures ellipsometric phase shift using focused laser light and Fourier analysis Need for translating stage Measured 0.6 nm changes Future work suggests using a CCD for higher throughput measures reflectivity changes around minimum wavelength using collimated monochromatic XE lamp-light Need for specially coated substrates Measured 1.4 +/- 0.2 nm Future work suggests using a laser source and focused light for greater resolution OI-RDRIDO

25. The end!