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TRANSCRIPT
Lara Gamble
University of Washington
Department of Bioengineering
Bi+
X-ray
PO3-
C5H4N3-
Si-
Si
© Lara Gamble (2011)
Surface Chemical State Imaging Analysis of DNA Microarrays
Objec-ves:
• Fluorescence imaging to determine DNA spot (Cy3) uniformity
• Imaging ToF-‐SIMS to determine surface distribuBon of different chemical species
2-‐3 seconds
Glass Slide
H2O
Glass Slide
nanoliter drop of
DNA
Dried DNA film
150µm
Glass Slide
↑ DNA concentra-on ↑ Ionic strength × Equilibrium
print pin or peizo printer
© Lara Gamble (2011)
Commercial CodeLink™ microarray slides (Amersham), and Slide H Schott Nexterion N-hydroxyl succinimide (NHS): active ester, amine-reactive
5’Cy3-‐C6 -‐CTG
AACG
GTAG
CATCTTGAC-‐C
6 -‐NH2
DNA ImmobilizaBon Chemistry on Commercial Polymer Slides
© Lara Gamble (2011)
Surface Chemistry of Commercial Slides as Determined by X-‐ray Photelectron Spectroscopy (XPS)
CodeLink™ (Amersham)
OptArray™ (Accelr8)
Si2
p S
i2s
C1s
N1s
C
a2p
O1s
Na1
s
Composition from survey scan
J. Am. Chem. Soc. 129 (30), p9429-9438, 2007 © Lara Gamble (2011)
Surface Chemistry of Commercial Slides as Determined by XPS
CodeLink™ (Amersham)
OptArray™ (Accelr8)
C-H, C-C
C-N, O-C-O C=O,
N-C=O Acrylamide based
Poly ethyleneglycol (PEG) based
J. Am. Chem. Soc. 129 (30), p9429-9438, 2007 © Lara Gamble (2011)
DNA ImmobilizaBon Chemistry on Commercial Polymer Slides
OH
O
O
O
OO
Hz
O
OO
Hx y
Tween20TM
OH
ON
O
N-Lauroyl Sarcosine (Sarcosyl)
CH2OHHOH2CCH2OHNH2
Tris(hydroxymethyl)aminomethane
SOOOO
Sodium Dodecyl Sulfate (SDS)
ONa
O OH
ONa
O
ONaO
Sodium Citrate
Prin-ng
Blocking
Washing NaCl
Sodium Chloride
*NESAC/BIO, University of Washington Seattle, WA. U.S.A.
© Lara Gamble (2011)
DNA microarray fluorescence
40 µM DNA probe conc.
25% Cy3 75% 100%
+ Fast and widely available
+ High resoluBon (5µm)
− Non-‐quanBtaBve
− Spot uniformity, reliability
− Spot bleeding into matrix
ObjecBves:
• Fluorescence imaging to determine DNA spot (Cy3) uniformity
• Imaging ToF-‐SIMS and staBsBcal analysis to determine surface distribuBon of different chemical species corresponding to the fluorescence non-‐uniformity
highest intensity -> Red lowest intensity -> Blue
Array fabricaBon • Ink jet, contact pin • Feature size ~100 microns diameter
© Lara Gamble (2011)
Slide prinBng
C6-‐oligo-‐3’ C6-‐NH2
Three different DNA concentra-ons: 10μm, 20μm, 40μm Four different Cy3 labeled % for each concentra-ons: 0%, 25%, 75%, 100% Ten replica-ons of each spot.
Volume size, 333 pL and spot size ~315 µm
Examine distribu-on of chemical species within DNA microarray spots
© Lara Gamble (2011)
Effects of immobilizing Probe mixtures containing different amounts of Cy3-labeled probe
Scanned microarray image (ScanArray Express, Perkin Elmer) Spots printed at 400 um center to center
10 uM print 20 uM print 40 uM print
1%
5%
10%
25%
50%
75%
100%
% Cy3 Labeled Probe
Hybridized with 1 uM Target (99:1 unlabeled:Cy5 labeled)
Dup
licat
e sp
ots
0%
1200 um
Scanner Image showing 1 array
© Lara Gamble (2011)
Time of Flight Secondary Ion Mass Spectrometry (ToF)-‐ SIMS
• Ultra High Vacuum (UHV) • Primary ion impact causes collision cascade • >99% ejected molecules are neutral, <1% are ions
ION-TOF ToF-SIMS 5-100:
• 25 kV Bi at 45° (Bi3+) • Analysis (Imaging) beam • High spatial resolution (<
1µm)
• 10 kV C60 at 45° (C60+ and C60
++) • High sputter yield • Sample etching
10
http://www.iontof.com/technique-sims-IONTOF-TOF-SIMS-TIME-OF-FLIGHT-SURFACE-ANALYSIS.htm
© Lara Gamble (2011)
SIMS imaging
• Image resoluBon 128x128 pixel -‐> 16384 spectra
• Use Maximum AutocorrelaBon Factor (MAF) analysis to find the peaks corresponding to the largest variance between the different spectra.
400x400µm 128x128 pixels
Bi3+ For a SIMS image each pixel is a spectra
Na2PO3+ PO3
-
© Lara Gamble (2011)
NegaBve Ion ToF-‐SIMS ROI Spectra from DNA and Substrate Regions
Total cou
nts
PO2 (63)
PO3 (79)
Ade-‐H (134)
Cyt-‐H (110)
Gua-‐H (150)
Thy-‐H (125)
Total cou
nts
DNA
Substrate
Total ion image
m/z m/z
m/z m/z
50µm
J. Am. Chem. Soc. 129 (30), p9429-9438, 2007 © Lara Gamble (2011)
ToF-‐SIMS Analysis of Microarray Spot Uniformity Field of view: 1.5 mm x 1.5 mm
Si-‐ PO-‐
Comp. probe (hybridized)
Non-‐comp. probe
Non-‐complementary probe
Complementary probe (hybridized)
PO2-‐ PO3
-‐ C5H4N3-‐
(adenine-‐H) C5H7N2O2
-‐
(thymine-‐H) C5H4N5O
-‐
(guanine-‐H) C4H4N3O
-‐ (cytosine-‐H)
PO2-‐ PO3
-‐ C5H4N3-‐
(adenine-‐H) C5H7N2O2
-‐
(thymine-‐H) C5H4N5O
-‐
(guanine-‐H) C4H4N3O
-‐ (cytosine-‐H)
Field of view: 200 µm x 200 µm
Imaging parameters:
• IONTOF IV • Bi+ primary ion source
• Pulsed 25 keV primary ion beam at 1.3 pA
J. Am. Chem. Soc. 129 (30), p9429-9438, 2007 © Lara Gamble (2011)
10 uM print Probe Target
20 uM print Probe Target
40 uM print Probe Target
0%
1%
5%
10%
25%
50%
75%
100%
Scale Bar 100 um
% C
y3 L
abel
ed P
robe
Hybridized with 1 uM Target (99:1 unlabeled:Cy5 labeled)
Corresponding fluorescent data
© Lara Gamble (2011)
ToF-‐SIMS Indicate Similar Molecular Species Found on Ring Structure and Damaged Regions
SiH- Si- PO3- SiCH- CHS-
SiO2- SiHO2
- SiO3- CHSO2
- SiCH3O-
Field of view: 200 µm x 200 µm Damages on non-comp. probe spots
J. Am. Chem. Soc. 129 (30), p9429-9438, 2007 © Lara Gamble (2011)
Maximum Autocorrela-on Factors (MAF)
• Find linear combinaBon of peaks that
– Maximize variance in image
– Minimize variance between neighboring pixels
• Improves image contrast
• Typically provides beger separaBon of chemical consBtuents
• Scaling independent
10 µM Cy3 25%
Total ion Intensity
Fluorescence Intensity
© Lara Gamble (2011)
Concerns
25%
75%
100%
• Spot size “changes” with concentraBon of DNA
• Type of spoger ‘magers’
• Results differ with different substrates
• The non-uniformity is probably due to the rapid drying of the droplet rather then separation of labeled and unlabeled DNA on this surface.
• DNA purity?
Σ DNA Σ Cy3 PO2
© Lara Gamble (2011)
Acknowledgments
• University of Washington, NESAC/BIO
• Prof. Dave Castner, Dr. Líney Árnadóir, Dr. Nicolas Vandencasteel
• University of Utah (Prof. Dave Grainger) • Greg Harbers, Shaun Bevers and Archana Rao
prin-ng and fluorescence data
• Funding: NIH Grant EB-‐002027 (NIBIB) and NIH Grant EB-‐001473
© Lara Gamble (2011)