LaraGamble$$ - Max Planck Societyweb.evolbio.mpg.de/ploenworkshop/presentations/LaraGamble.pdf ·...

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


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


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


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.


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


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


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


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


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

-


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


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


10 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


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


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


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


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


LaraGamble$$ - Max Planck Societyweb.evolbio.mpg.de/ploenworkshop/presentations/LaraGamble.pdf ·...

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