Label Free Biomolecular Detection using Ellipsometric principles: Two Methods Jeremy Colson.
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Transcript of Label Free Biomolecular Detection using Ellipsometric principles: Two Methods Jeremy Colson.
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Label Free Biomolecular Detection using Ellipsometric principles: Two Methods
Jeremy Colson
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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.
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Outline
1. Reflection coefficients. Ellipsometric ratio2. Method 1: OI-RD
1. Calculations2. Setup3. Results
3. Method 2: RIDO1. Setup2. Theory3. Results
4. Conclusion
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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
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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:
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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)
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Calculation Verification 1
p s 0 0
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Calculation Verification 2
0)Im( sp
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Calculation Verification 3
p s j40
1 2s sin coss 0 s cos2 0 sin2
d s d 0
d
d
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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
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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)
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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 )
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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
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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.
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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
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Reflection interferometric detection
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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)
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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
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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
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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.
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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
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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.
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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
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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
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The end!