Label Free Biomolecular Detection using Ellipsometric principles: Two Methods
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Transcript of Label Free Biomolecular Detection using Ellipsometric principles: Two Methods
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.
Outline1. 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 j 401 2s sin cos
s 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 j 401 2s sin cos
s 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 j 401 2s sin cos
s 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’dFor 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!