Label Free Biomolecular Detection using Ellipsometric principles: Two Methods

Jeremy Colson

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.

Outline

1. Reflection coefficients. Ellipsometric ratio2. Method 1: OI-RD

1. Calculations2. Setup3. Results

3. Method 2: RIDO1. Setup2. Theory3. Results

4. Conclusion

Reflection Coefficients and the Ellipsometric Ratio

rp0 N2 cos1 N1 cos2

N2 cos1 N1 cos2

;rs0 N1 cos1 N2 cos2

N1 cos1 N2 cos2

rp rs tane j

Total Reflection Coefficient for a Film on a Substrate:

•Phase change for one trip through film:

˜ N 1

˜ N 2

˜ N 3

1

2

3

2 d

N 2 cos2

#1

# 2

# 3

•Adding partial waves

R r12 r23e

j 2

1 r12r23e j 2

r21r23e j 2

converging term:

OI-RD Calculations

p rp rp 0 rp 0

p s 0 0For

d

p s j40

1 2s sin coss 0 s cos2 0 sin2

d s d 0

d

d

)(csc2)Re(,)Im( 000 spsp

It has been shown that

* A.Wong and X.D.Zhu.Appl. Phys. A 63, 1 (1996)

Calculation Verification 1

p s 0 0

Calculation Verification 2

0)Im( sp

Calculation Verification 3

p s j40

1 2s sin coss 0 s cos2 0 sin2

d s d 0

d

d

IO-RD Calc’s: 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)

p s j40

1 2s sin coss 0 s cos2 0 sin2

d s d 0

d

d

0)Im( sp

OI-RD Experimental Setup

1. p-polarized He-Ne laser (632nm)2. Photoelastic modulator oscillates

polarization (50kHz)3. Pockels cell to adjust phase

difference4. Lens focuses beam (3µm)5. Reflection (45°) and

recollimation6. Rotatable analyzer converts

oscillating polarization to oscillating intensity

7. Photodiode detects I(t)

OI-RD Data Collection/Calibration Procedure

First and second harmonics analyzed with lock-in amplifiers

Reflection off bare substrate: I(2Ω) =0 with analyzer I(Ω) = 0 with Pockels Cell

Subsequent scans: I(Ω) = phase shift ~ Im(∆p-∆s )

I(2Ω) = Re(∆p-∆s )

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

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.

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

Reflection interferometric detection

RIDO Experimental Setup

1. S: 450-W Xe lamp monochromatized to ~1 nm bandwidth

2. P: s-polarizer3. A: ~5 mm apertures (enforces collimation)4. Incident light at 70.6°5. D: CCD detector (Roeper Scientific)

Theory

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

r12 r23 exp( 2 j) 1 r12r23 exp( 2 j) 20

r12 r23; 2

1min sin 1 (n32 n2

4 /n12) (n1

2 n32 2n2

2) 1/ 2

2 d

n2 cos2

dmin / (1/4)(n22 n1

2 sin2 1)1/ 2

Theory cont’d

For air/SiO2/Si with 660 nm wavelength:

n1 = 1; n2 = 1.4563; n3 = 3.8251

1min 70.6; dmin / 0.2253

For ideal conditions (perfectly flat surface, collimated monochromatic light) reflectivity changes by > factor of 10 for .22 nm thickness change at min. wavelength

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.

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

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.

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

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-RD RIDO

The end!