printing high quality protein microarrays

Printing High Quality Printing High Quality MicroarraysMicroarrays

Todd [email protected]://arrayit.com

arrayit.comarrayit.com

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.

Printing High QualityPrinting High QualityProtein MicroarraysProtein 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!

The Methodology of PrintingTechnologies

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

What you need to do and why.

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

TeleChem/ArrayIt.comArrayIt.com

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

NonNon--Contact TypesContact Types

Perkin Elmer & others


To my knowledge, not used commercially

Very high heating of the sample makes it problematic

Difficult to change samples


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

Well made ceramic tips

Different tips for different spot sizes and volumes

NonNon--Contact TypesContact 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 Contact Printing –– Pin & RingPin & 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 Pinsand Quillsand Quills

Tweezers / 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

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 PinsMicro Spotting Pins

•Defined sample uptake (0.25, 0.6 or 1.25 ul)•Sample at end of flat tip•Substrate pulls off drop

Stealth Micro Spotting Device Stealth Micro Spotting Device SubSub--nanoliter Vol. Dispensingnanoliter Vol. Dispensing

PTO# 6,101,946Digitally controlled manufacturing 355X

Spotting Sequence…Spotting Sequence…

++ 2 Micron Tolerance

Mechanically identical parts Mechanically identical parts perform identical tasksperform identical tasks

Typical ResultsTypical Results

Spot #1

Spot # 200

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

ArrayItArrayIt Stealth 3 PinStealth 3 Pin

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

Printing High QualityPrinting High QualityProtein MicroarraysProtein Microarrays

2. Sample Preparation

Contaminates spotted Contaminates spotted sample…sample…

Prohibit samples from immobilizing on the microarray printing servicesProhibit interaction between array elements and probesCause background noiseCan 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

– 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 BetterBetter

Spotting BufferSpotting Buffer

Microplates and Samples384 round wells, not 96 wells

better for avoiding evaporationRigid polypropylene constructionV or U bottom shaped wells3-15 microliters of sample per well

Polypropylene Polystyrene

Sample

Does not bind DNA Binds DNA


3. Robotics

A good microarrayer has…A good microarrayer has…

Accuracy and repeatability on the micron levelComputer controlled GUI for easy programming and sample trackingGood wash/dry station between sample changes to eliminate cross contamination between samplesHumidity and temperature control in a closed “cleanroom” level positive pressure environment

A good example…A good example…

ChipWriter Pro from Bio-Rad

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


Avoiding Sample CarryoverAvoiding Sample CarryoverUse 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

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

3D (absorption) vs. 2D (covalent) Surfaces3D (absorption) vs. 2D (covalent) Surfacesin general…in 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 proteinswww.schleicher-schuell.com

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

Angstroms

Inte

ns i

t y S

cale 50.0

25.0

0.0

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

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


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 storeReplace old ceiling tilesKeep arrayers away from air vents or add filters to incoming airBuy an arrayer with good environmental controlHave 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

ClassClass--100 Clean Room 100 Clean Room EnvironmentEnvironment

DustDust--Free Free

-- Air filtered by Air filtered by ULPA FiltrationULPA Filtration

Precisely ControlledPrecisely Controlled-- HumidityHumidity--Temperature Temperature ––


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

Miragene Inc.unpublished

MacBeath & Schrieber, Science, 289:1760, 2000

Schleicher-Schuellunpublished

Stears, et al. Nature Med 2003.

Zhu, H. et al. (2001)

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

LipidsLipids

DNADNA

Small moleculesSmall moleculesCarbohydrates

printing high quality protein microarrays

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