Printing High Quality Microarrays

1. arrayit.com Printing High Quality Microarrays Todd Martinsky todd@arrayit.com http://arrayit.com

2. arrayit.com Outline • Company background • In the media • Key personnel • Established credibility • Making the perfect microarray • It’s really a science, not an art • Keys 1 through 5 • Conclusions

3. arrayit.com • Privately held company • Financially strong and profitable • Fastest Growing Biotech in the America • Microarray Technologies • First company to create microarray products • Superior technical expertise • Recognized Leader in the field • 10 year anniversary Nov. 1, 2003

4. 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.

5. arrayit.com Award Winning Service & Quality Silver Medal From DOD

6. Largest Circulationin the United States. • 12 Companies mentioned • 2 mentioned twice • TeleChem & Affymetrix

7. Featured on NOVA

8. TeleChem /arrayit.com How We Develop Products Combine engineering principles with biological expertise (Parallel Gene Analysis Methodology) to systematically develop microarray products and services.

9. 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?

10. In 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

11. In 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

12. 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)

13. That’s me there • 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)

14. Summary by Month Month Daily 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 Totals 65486712 195099 1254415 5550418 8232338 Protocols on the web – arrayit.com

15. Making the Perfect Microarray Our golden rule, “If there is a variable in your system, control it.”

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

17. Key Factors to Control • Micro fluidic printing technology • Robotics (including wash/dry station) • Sample preparation • Surface chemistry • Environment • If you’ve got a quality problem, I can guarantee it’s in 1 of these 5 areas.

18. Printing High Quality Protein Microarrays 1. Printing Mechanisms We should appreciate the fact that 1 picoliter is to 1 liter as 1 cm is to 13 round-trips to the moon!

19. The Methodology of PrintingTechnologies Our 12 Rules…..being published later this year by Kluwer The principles that determine how spotting technologies are used and interpreted. What you need to do and why. TeleChem/ArrayIt.com

20. 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

21. 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

22. Two Main Types • Consider 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

23. Non-Contact Types Perkin Elmer & others

24. Non-Contact Types To my knowledge, not used commercially Very high heating of the sample makes it problematic Difficult to change samples

25. Non-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

26. Non-Contact Types Change 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

27. Non Contact IMIT’s TopSpot Uses an Piezo actuator and micro fluidic channels. Industrial level manufacturing of the same array

28. Contact 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

29. 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 Split Pins and Quills Split Pins (Many) Tweezers / Quills (Schena et al.,1995) • Variable sample uptake • Forms a meniscus • Tapping expels sample

30. Patented Micro Spotting Pins by TeleChem • 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) Micro Spotting Pins • Defined sample uptake (0.25, 0.6 or 1.25 ul) • Sample at end of flat tip • Substrate pulls off drop

31. Stealth Micro Spotting Device Sub-nanoliter Vol. Dispensing PTO# 6,101,946 Digitally controlled manufacturing 355X

32. Spotting Sequence…

33. + 2 Micron Tolerance Mechanically identical parts perform identical tasks

34. Typical Results Spot #1 Spot # 200 Cy3 Labeled oligo in Micro Spotting Solution-1 equal spot sizes, equal signal intensities ArrayIt Stealth 3 Pin

35. Diameter Circularity Uniformity Average 113.2 0.95 1.00 STD 4.2 0.01 0.00 CV 0.04 0.01 0.00 QuantArray analysis software (Packard Biosystems) data for 300 spots. Analysis of Typical Results Note:When the key elements 1-5 are controlled properly

36. Printing High Quality Protein Microarrays 2. Sample Preparation

37. Contaminates in 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

38. PCR PurificationMembrane vs. ETOH Precipitation Data

39. PCR and Fluorescent Probe Purification

40. Bad Better Spotting Buffer Qualities of a good spotting 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

41. 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 U Sample Polypropylene Polystyrene Does not bind DNA Binds DNA

42. Making the Perfect Microarray 3. Robotics

43. 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

44. A good example… Self contained environmental (humidity) controlled chamber to clean room level quality dynamicdevices Oasis TeleChem/ArrayIt.com

45. Avoiding Sample Carryover Use multiple wash/dry cycles, never dry the printing mechanism until the last wash cycle is complete! Is the job of the wash/dry station on the microarrayer

46. Minimum Software Requirements • 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!!!!!

47. Easy Programming Example

48. Personal Microarray System ~1000 samples every 2 hours over 14 substrates may be high enough throughput?

49. Making the Perfect Microarray 4. Sample Immobilization

50. 3D (absorption) vs. 2D (covalent) Surfacesin general… • 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 proteins www.schleicher-schuell.com