Microarray Feature Uniformity

1
Electrostatic Printing of Composite Features for
Highly Uniform DNA Microarrays and Other
Innovations in Microarray Technology
P. Dextras, K. Guggenheimer, J. Thompson and A.
Marziali Department of Physics and Astronomy,
University of British Columbia, Vancouver, BC,
Canada

• A reverse voltage is applied across the capillary
  to flush out any unbound target. The reverse
  potential is kept low enough to prevent
  dissociating the hybridized DNA molecules.
• After removing the unbound target molecules the
  end of the capillaries are imaged with a laser
  confocal scanner.
• After scanning fresh LPA is pumped into the end
  of the capillary and another assay can be
  performed.

• 3.2. Composite feature printing methodology
• The prototype is currently used to print a 50 ?M
  solution of a 25-mer oligonucleotide with a 5
  6-FAM fluorescent tag. The current aim is to
  determine the optimum voltage, pulse width, spot
  spacing and time between prints for maximum
  uniformity.
• Fluorescence uniformity is measured by imaging
  the slide on an Applied Precision arrayWoRx
  scanner with a resolution of 4 ?m. The physical
  morphology is determined using a scanning
  electron microscope.
• Square features are currently printed in order to
  simplify the LabView control software however,
  any arbitrary shape could be printed.
• Originally, features were built up by printing in
  a raster pattern, but this resulted in mass
  transfer of solution towards the leading edge of
  the feature. A higher uniformity can be achieved
  by first printing the odd rows of the feature and
  then filling in the even rows.

• GenomeBC Technology Development Team
• GenomeBC is a non-profit organization funded by
  GenomeCanada to fund and promote Genomics
  research in British
• Columbia. The Technology Development Team was
  established at UBC to provide shared technology
  development
• and engineering support for GenomeBC projects.

• Our current focus is to investigate sources of
  technical error in microarray methodologies and
  to develop
• novel microarraying technologies. The two main
  projects being pursued are
• Electrostatic Printing of Composite Features for
  Highly Uniform DNA Microarrays
• Using a pulsed field droplet spotter, we are
  developing a new method for printing highly
  uniform microarray features.
• Replaceable Gel-based Hybridization Assay
• This device performs DNA hybridization
  experiments inside glass capillaries with both
  probe and target in free solution.
• For information on other current technology
  development projects, refer to
  www.physics.ubc.ca/andre/

A
B
D
C
(A) Schematic of an oligo bound to an LPA
molecule. (B) End of a capillary exposed to
target solution (C) Electrokinetic injection of
target molecules. (D) Reverse voltage applied to
remove unbound target.
4.3 Current Progress A version of this device
with a single capillary and a two colour laser
confocal scanning system capable of imaging cy3
and cy5 has been built. Initial single colour
tests with 6-FAM tagged oligos have shown that we
can distinguish between an oligo perfectly
matched to the probe and one with a complete
mismatch (see below).

• Microarray Feature Uniformity
• Printing with contact devices such as tungsten
  pins often results in concentration and
  morphology variation.
• Fluorescence can be quenched or change emission
  wavelength based on the local concentration of
  dyes.
• Red/ Green ratios may vary from pixel to pixel
  (Brown, C. Goodwin, P. Sorger, P. 2001. Image
  metrics in the statistical analysis of DNA
  microarray data. PNAS 98 (16) 8944-8949.)
• Our goal is to develop a method of printing
  features which reduces non-uniformity in
  microarray data.

Scanning electron microscope image (above left),
and array scanner fluorescence image in 2D
(center) and 3D (right).

• 3.3 Future direction of the project
• We plan to apply a conventional surface
  chemistry to the ITO-coated slides to covalently
  bond probe molecules for
• hybridization experiments.
• Multiple independently-addressable
  capillaries will be integrated into a single
  print head for high-speed printing of
• large microarrays.

Fluorescence image (above) and 3D plot (left)
showing morphology variation in pin- printed
microarray features.
Fluorescence signal from a complementary oligo
probe (left-top) and a non-complementary oligo
probe (left-bottom). A schematic of a single
capillary version of this device (right)

• Electrostatic Printing of Composite Features for
  Highly Uniform DNA Microarrays
• 3.1 Pulsed field droplet spotter

• 4.4 Advantages over slide based arrays
• Printing the printing step has been reduced to
  simply pumping fresh LPA into the end of a
  capillary
• Imaging imaging is simplified because the
  location of the spots is always fixed, and the
  morphology of the spot is always the same.
• Hybridization Speed because target is
  electrokinetically injected into the capillary
  you dont have to wait for targets to diffuse to
  spots, also steric hindrance is less of a
  problem.
• 4.5 Future Work
• Optimize the single capillary device for
  distinguishing single base pair mismatches.
• Expand to a multiple capillary device, with the
  ultimate goal of incorporating 96 or 384
  capillaries in a single device. Here capillaries
  would be close packed at the hybridization end,
  and the other end would be re-arrayed into
  microtiter plate format, where each well in a
  microtiter plate would contain a different probe
  oligonucleotide.

• Replaceable Gel-based Hybridization Assay
• 4.1 Device Overview
• We are currently developing a device for
  performing gel-based hybridization assays, with
  replaceable linear polyacrylamide (LPA) inside of
  a glass capillary. The goal of this work is to
  develop a device for performing extremely high
  throughput hybridization assays on a relatively
  small number (hundreds) of SNPs or genes.

• A non-contact printing device that is able to
  deliver small liquid volumes onto conductive
  substrates (Yogi, et. al. 2001. On Demand
  Droplet Spotter for Preparing Pico- to-Femtoliter
  Droplets on Surfaces. Anal. Chem. 73 (8)
  1896-1902).
• Prints electrostatically when a voltage pulse is
  applied between the solution in the capillary and
  the substrate. A clear, electrically conductive
  printing surface is required (currently using an
  indium tin oxide coating on glass).
• The prototype system can print spots as small as
  2µm in diameter. This makes it possible to
  construct large (100µm) features from
  overlapping spots resulting in a high overall
  uniformity.

• 4.2 Device Operation
• The first step in the hybridization assay is to
  fill a capillary with LPA in which acrylamide
  modified probe
• oligonucleotides have been incorporated into the
  polymer chains
• Next the end of the capillary is exposed to a
  solution of tagged target DNA.
• A voltage is then applied across the capillary to
  electrokinetically inject target DNA into the
  capillary, here complementary target DNA will
  hybridize to the bound probe molecules.

Acknowledgements We thank GenomeBC for financial
support. We also thank Scott Tebbutt and the
iCAPTUR4E Center, Colleen Nelson, Jeff Zeznik
and the Jack Bell Research Centre for their
contributions.