printing high quality microarrays todd martinsky todd@arrayit.com

Printing High Quality Printing High Quality MicroarraysMicroarrays

Todd Martinskytodd@arrayit.comhttp://arrayit.com

arrayit.coarrayit.comm

Outline Outline Company backgroundIn the mediaKey personnel

– Established credibilityMaking the perfect microarray

– It’s really a science, not an art– Keys 1 through 5

Conclusions

arrayit.coarrayit.comm

• Privately held company Privately held company

• Financially strong and profitableFinancially strong and profitable

• Fastest Growing Biotech in the AmericaFastest Growing Biotech in the America

• Microarray TechnologiesMicroarray Technologies

• First company to create microarray productsFirst company to create microarray products

• Superior technical expertiseSuperior technical expertise

• Recognized Leader in the fieldRecognized Leader in the field

•10 year anniversary Nov. 1, 200310 year anniversary Nov. 1, 2003

arrayit.coarrayit.comm

• Developed First Micro Spotting Pins for

Microarray Manufacturing

-Intellectual property from 1997

• Most Widely used Microarray Technology in the

world Patent # 6101946

• World leader in microarray consumables

- Glass substrates, Pins, Hybridization

cassettes, spotting solutions, sample prep

kits, etc.

Award Winning Service & QualityAward Winning Service & Quality

Silver Medal

From

DOD

arrayit.coarrayit.comm

Largest Circulationin the United States.

12 Companies mentioned

2 mentioned twice TeleChem &

Affymetrix

Featured on NOVAFeatured on NOVA

How We Develop ProductsHow We Develop Products

Combine engineering principles with Combine engineering principles with biological expertise (Parallel Gene biological expertise (Parallel Gene Analysis Methodology) to systematically Analysis Methodology) to systematically develop microarray products and develop microarray products and services.services.

TeleChem / arrayit.comarrayit.com

Parallel Gene Analysis Methodology– What you need to do and why, to complete

the microarray lifecycle. – Keep in simple, don’t over-engineer– Complicated systems and schemes break

down and are typically expensive Critical thinking Customer Feedback End Result: products, methods &

protocols – Derived from our integrated In-House

engineering and biology expertise Who are these people?

In House Engineering ExpertiseIn House Engineering Expertise

Richard Martinsky: Director of Engineering– Designed & Invented:

Hybridization Cassette Microarray Wash Station Stealth & Chipmaker Micro Spotting Devices

– Process Engineering: Substrate manufacturing Microarray Manufacturing

– Wash/Dry stations for Pins– Microarray Scanners– Microarray Robotics

In House Microarray ExpertiseIn House Microarray Expertise

• Mark Schena, Ph.D., Visiting Scholar

• Entire Microarray Industry based on first paper:

- Schena et al. Science. 270:467. 1995.

• Editor of books on microarray Technology - (Oxford University Press, Biotechniques publishers)

• Wrote the textbook on microarrays - (Wiley & Sons, Largest publisher of textbooks in the world)

• Recognized World Expert on Microarray Technology

More Expertise…More Expertise…

Robin L. Stears, Ph.D. Senior Scientist/Director of Microarray Technology

– Utilized First commercially available microarryer & Scanner -

1997 (Laboratory of Dr. S. Gullans, Harvard Medical School)

– Developed Genisphere 3DNA microarray detection method (Stears et. Al, Physiological Genomics, 2000)

– Head of Transcriptional Profiling Team, Affymetrix platform,

Aventis Pharmaceuticals (2000-2001, Cambridge Genomics Center, MA)

• Recognized leader with technological innovation, intellectual property

• Top names in the field-• Mark Schena, Ph.D• Robin L. Stears, Ph.D.

• Creating turn-key solutions for DNA Microarrays since 1996 (ArrayIt Brand Products)

That’s me there

Protocols on the web – Protocols on the web – arrayit.comarrayit.com

Summary by Month

MonthDaily Avg Monthly Totals

Hits Files Pages Visits Sites KBytes Visits Pages Files Hits

Jan 2003 32472 24052 4290 648 3741 2356350 6485 42907 240527 324726

Dec 2002 29811 20335 4644 594 8890 6346647 18414 143979 630386 924152

Nov 2002 23953 16355 3427 411 7142 5439463 12338 102820 490659 718613

Oct 2002 27323 18979 4415 670 9638 7636378 20776 136874 588362 847016

Sep 2002 24863 16711 4399 643 8107 6593075 19300 131996 501352 745900

Aug 2002 9086 6068 1515 269 3766 2356590 8368 46976 188134 281672

Jul 2002 21104 13970 2917 531 7157 5451430 16483 90457 433090 654224

Jun 2002 23863 16185 4306 663 8284 5995133 19893 129206 485569 715902

May 2002 26011 16551 4066 688 8227 6258849 21331 126054 513103 806367

Apr 2002 26316 17642 3565 624 8087 6204948 18738 106965 529267 789480

Mar 2002 23505 15509 3302 566 7917 5449506 17574 102367 480808 728656

Feb 2002 24843 16755 3350 549 7243 5398343 15399 93814 469161 695630

Totals6548671

219509

9125441

5555041

8823233

8

Making the Perfect Making the Perfect MicroarrayMicroarray

Our golden rule,

“If there is a variable in your system, control it.”

This information is based on 8 years experience providing technical support for microarray manufacturing….

Key Factors to ControlKey Factors to Control

1. Micro fluidic printing technology

2. Robotics (including wash/dry station)

3. Sample preparation

4. Surface chemistry

5. Environment

• If you’ve got a quality problem, I can guarantee it’s in 1 of these 5 areas.

1. Printing Mechanisms

Printing High QualityPrinting High Quality Protein Microarrays Protein Microarrays

We should appreciate the fact that 1 picoliter is to 1 liter as 1 cm is to 13 round-trips to the moon!

Our 12 Rules…..being published later this year Our 12 Rules…..being published later this year by Kluwerby Kluwer

The principles that determine how spotting technologies are used and interpreted.

What you need to do and why.

TeleChem/ArrayIt.comArrayIt.com

The Methodology of Printing Technologies

12 rules continued…12 rules continued…1. Print uniform spots measured in microns

2. Print individual spots in regular array patterns that can be tracked by computer

3. Easy to implement

4. Cost effective/affordable

5. Print without damaging the sample or surface chemistry

6. Saturate the immobilization surface chemistry at each spot location

12 rules continued…12 rules continued…7.  Amenable to high and low density

8.  Change spot sizes and sample volumes easily

9.  Load and deliver a specific amount of sample each time

10. Easy to fix and maintain, with no special tooling or tech visits required

11. Compatible with a variety of scientific applications

12. Print multiple samples, multiple times on multiple substrates with one low volume loading of sample

Two Main TypesTwo Main TypesConsider the efficient use of sample when making your choice!

Contact

Best for high numbers of samples over many substrates

Non-contact

Low numbers of samples over many, many substrates

Non-Contact TypesNon-Contact Types

Perkin Elmer & others

Non-Contact TypesNon-Contact Types

To my knowledge, not used commercially

Very high heating of the sample makes it problematic

Difficult to change samples

Non-Contact TypesNon-Contact Types

Best for low numbers of samples and high numbers of spots.

Well made ceramic tips

Different tips for different spot sizes and volumes

Non-Contact TypesNon-Contact TypesChange delivery volume by “firing” multiple times in the same spot location

Spot size on par with Pin spotting

Typically slower than Pin spotting since commercial systems are limited to 4-8 delivery nozzles

Glass capillaries

Perkin Elmer Type

Non ContactNon Contact

IMIT’s TopSpot Uses an Piezo actuator and micro fluidic channels.

Industrial level manufacturing of the same array

Contact Printing – Pin & RingContact Printing – Pin & Ring

Advantages:

Multiple prints with 1 load

Consistent and reliable

Disadvantages:

Fixed number of Pins (4)

Large uptake volume

Low delivery vol. per spot

Spring Loaded Pins

No flexibility to change spot size

Complex actuators not easy to fix (heat up during long runs)

Large source plate vol. 96 well plates

No longer supported by Affymetrix

What sticks to the tip of the pin as it passes through the ring defines the amount of sample delivered

Split PinsSplit Pins and Quills and QuillsTweezers / QuillsTweezers / Quills

(Schena et al.,1995)(Schena et al.,1995)Split PinsSplit Pins

(Many)(Many)

•Variable sample uptake

•Forms a meniscus

•Tapping expels sample

Advantages:

Multiple prints with 1 low volume of load

Patent owned by Incyte, but not commercialized by them

Flexible to change # of pins used only

Can be replaced by user

Disadvantages:

Spring loaded (force on tips) Tapping force to expel sample wears them out quickly / variable deposition of sample

Tip tolerances uneven (ref., Brown patent)

No flexibility to change spot size

Mistakes are expensive

Patented Patented Micro Spotting Pins by TeleChemMicro Spotting Pins by TeleChem

Micro Spotting PinsMicro Spotting Pins

•Defined sample uptake (0.25, 0.6 or 1.25 ul)

•Sample at end of flat tip

•Substrate pulls off drop

Advantages:

Multiple consistent prints with 1 low vol. load

Patented and commercialized by the same organization with compatible consumables

Flexible to change # of pins and spot size

Easy to fix

Widely used

Tight tolerances and quality control

Durable (under the right motion parameters)

Low volume of sample in source plate (96 & 384 well)

Stealth Micro Spotting Device Stealth Micro Spotting Device Sub-nanoliter Vol. DispensingSub-nanoliter Vol. Dispensing

355XPTO# 6,101,946Digitally controlled manufacturing

Spotting Sequence…Spotting Sequence…

++ 2 Micron Tolerance

Mechanically identical parts Mechanically identical parts perform identical tasksperform identical tasks

Typical ResultsTypical Results

Spot # 200

Cy3 Labeled oligo in Micro Spotting Solution-1equal spot sizes, equal signal intensities

ArrayItArrayIt Stealth 3 Stealth 3 PinPin

Spot #1

Analysis of Typical ResultsAnalysis of Typical Results

QuantArray analysis software (Packard Biosystems) data for 300 spots.

  Diameter Circularity Uniformity

Average 113.2 0.95 1.00

STD 4.2 0.01 0.00

CV 0.04 0.01 0.00

Note:When the key elements 1-5 are controlled properly

2. Sample Preparation

Printing High QualityPrinting High Quality Protein Microarrays Protein Microarrays

Contaminates in Contaminates in spotted sample…spotted sample…

Prohibit samples from immobilizing on the microarray printing services

Prohibit interaction between array elements and probes

Cause background noise

Can clog pins and other printing mechanisms

Ruin spot morphology

PCR PurificationPCR PurificationMembrane vs. ETOH Membrane vs. ETOH

Precipitation DataPrecipitation Data

PCR and Fluorescent Probe PCR and Fluorescent Probe PurificationPurification

Spotting BufferSpotting Buffer

– Print even, small, round spots– Disperses the sample evenly within the spot– Promote sample binding to the array surface– Retard evaporation within the source plates– Dry evenly, perhaps not dry at all– Wash away easily – Optimize attachment– Dry down and re-suspend– Visual after spotting regardless of surface– Stabilize sample for long term storage

Qualities of a good spotting buffer:Qualities of a good spotting buffer:

BadBad Better Better

Microplates and Samples 384 round wells, not 96 wells

better for avoiding evaporation Rigid polypropylene construction V or U bottom shaped wells 3-15 microliters of sample per well

U UPolypropylene Polystyrene

Sample

Does not bind DNA Binds DNA

Making the Perfect Making the Perfect MicroarrayMicroarray

3. Robotics

A good microarrayer has…A good microarrayer has… Accuracy and repeatability on the micron

level

Computer controlled GUI for easy programming and sample tracking

Good wash/dry station between sample changes to eliminate cross contamination between samples

Humidity and temperature control in a closed “cleanroom” level positive pressure environment

Self contained environmental (humidity) controlled chamber to clean room level quality

TeleChem/ArrayIt.comArrayIt.comdynamicdevices Oasis

A good example…A good example…

Avoiding Sample CarryoverAvoiding Sample Carryover

Is the job of the wash/dry station on the microarrayer

Use multiple wash/dry cycles, never dry the printing mechanism until the last wash cycle is complete!

Minimum Software Minimum Software RequirementsRequirements

•Number of sample delivery mechanisms and the center-to-center spacing of said mechanisms (4.5mm or 9mm centers).

•The total number of samples to be printed

•Offsets relative to the substrate

•Number of replicates of each sample

•Center-to-center distance between spots

•Number of columns and rows

•Number of substrates/slides to be printed

•Wash/dry parameters for the printing mechanisms between printing cycles.

•Mapping!!!!!

Easy Programming ExampleEasy Programming Example

Personal Microarray Personal Microarray SystemSystem

~1000 samples every 2 hours over 14 substrates may be high enough throughput?

Making the Perfect MicroarrayMaking the Perfect Microarray

4. Sample Immobilization

Advantages of 3D (membranes, filters & gels)– High binding capacity

(absorption)

– Compatible with fluorescent, chemiluminescent, colorimetric, radioactive detection

– Longer history of use (comfort level for users)

– Less expensive labeling reagents and reading equipment (colorimetric)

14µm thick nitrocellulose-based coating. Electron micrograph image above, the uniform pore structure provides a large, 3-dimensional surface area for protein binding. The 3-dimensional surface quantitatively binds arrayed proteinswww.schleicher-schuell.com

3D (absorption) vs. 2D (covalent) Surfaces3D (absorption) vs. 2D (covalent) Surfacesin general…in general…

Advantages of 2D– Better defined spot morphology

(no diffusion)– Inherent lower background

fluorescence (glass)– High specificity– Non-porous surface (no place to

trap any contaminate in processing)

– Covalent and/or specific binding for more stringent processing conditions

Angstroms

Inte

nsit

y S

cale

50.0

25.0

0.0

3D (absorption) vs. 2D (covalent) Surfaces3D (absorption) vs. 2D (covalent) Surfacesin general…in general…

Homogenous distribution of capture reactive groups across the entire surface is critical for attaching the same amount of sample at each array location

The printing mechanism must saturate capture groups at each spot location, since what does not bind washes away in processing

Effects of Spotting Effects of Spotting Surface & MorphologySurface & Morphology

HomogenousHomogenous HeterogeneousHeterogeneous

2D, Amino Silane

2D, Aldehyde coupling2D, Aldehyde coupling

2D, Epoxy Coupling2D, Epoxy Coupling

Reacts with Primary amines, but does not require dehydration

Making the Perfect Making the Perfect MicroarrayMicroarray

5. Environment

Environmental Keys…Environmental Keys…

• Cleanliness• Temperature • Humidity• Clean rooms help

but aren’t necessary

Easy Ways “Clean Up”Easy Ways “Clean Up”

Hepa Air Filters from your local hardware store

Replace old ceiling tiles

Keep arrayers away from air vents or add filters to incoming air

Buy an arrayer with good environmental control

Have a dedicated microarraying environment

Note on HumidityNote on Humidity

Take it out of the room (work comfort) and add it to the arraying chamber (sample evaporation)

It is too difficult to control an entire room, but easy inside a “small” arraying chamber

Notes on GlovesNotes on Gloves

Avoid latex gloves that leave contaminating protein residue

Use powder free gloves only

Synthetic rubber

Class-100 Clean Room Class-100 Clean Room EnvironmentEnvironment

Dust-Free Dust-Free

- Air filtered by - Air filtered by ULPA FiltrationULPA Filtration

Precisely Precisely ControlledControlled

- Humidity- Humidity

-Temperature Temperature ––

TeleChem/ArrayIt.comArrayIt.com

When the key elements When the key elements are controlled…are controlled…

It works…

Microarray Resource Center™

http://arrayit.com/e-library/

1,965 total publications for "microarray"

Haab, et al, Genome Biology 2001 2(2): research 0004.1-0004.13

MacBeath & Schrieber, Science, 289:1760, 2000

Zhu, H. et al. (2001)

Stears, et al. Nature Med 2003.

Schleicher-Schuellunpublished

Miragene Inc.unpublished

Personal Microarray Core Facility Personal Microarray Core Facility - $30K instead of $150K- $30K instead of $150K

In development a complete

line of hardware,

software, and consumables

(colorimetric, single color, enzymatic labeling)

Microarrays:Microarrays:Universal Biochemistry PlatformsUniversal Biochemistry Platforms

PeptidesPeptides ProteinsProteins

Carbohydrates

LipidsLipids

Small moleculesSmall molecules

DNADNA