Protein Microarrays:Protein Microarrays:Approaches to PrintingApproaches to Printing

Steven Suchyta Ph. D.Steven Suchyta Ph. D.Applications ScientistApplications ScientistGenomic Solutions IncGenomic Solutions Inc..

Protein MicroarraysProtein Microarrays

• Immobilize proteins onto small surfaces (in large numbers)

• Using proven and existing technology – DNA microarrays

• Specialized protein technology

Protein MicroarraysProtein Microarrays

• Protein Microarray Workflow• Measure protein expression • Antibody binding• Screen & assess patterns of

interaction

Significance:Significance:

>50 citations in Genbank in 2005

Protein Microarray WorkflowProtein Microarray Workflow

• Important factors:• Printing – Focus of this talk• Surface -Substrate• Imaging

Optimizing protein microarray Optimizing protein microarray spottingspotting

• For visualizing spots for optimization – do not want to use costly protein.

• Red food dye (~5%) in the spotting buffer

• Fluoresces in the CY3 channel

Protein MicroarraysProtein Microarrays• Substrates:• 3 dimensional • Specialized for protein

applications:• Slide H - hydrogel (Schott

Nexterion)• FAST - nitrocellulose (Whatman) • Slide format:

Printing onto 3D substratesPrinting onto 3D substrates

Microgrid II Omnigrid Accent

Contact printing/split pins

Considerations for 3D surfacesConsiderations for 3D surfaces

• Avoid Damage to 3D surface –

Omnigrid System:

Contact of the pin to the surface can be precisely calibrated.

Z Coordinate of the pin tip as it touches the surface can be finely adjusted (2.5 micron resolution).

Microgrid System:

Soft Touch: greatly reduce the speed of travel of the print head before it reaches the slide surface.

100mm/s to ~1 – 4 mm/s

Considerations for 3D surfacesConsiderations for 3D surfaces

• Avoid Damage to 3D surface –

Minimizing Carry-over Minimizing Carry-over ContaminationContamination

• Composition and differing viscosity of various spotting solutions – PBS, Glycerol

• Minimize the amount of carry-over between samples

Minimizing Carry-over Minimizing Carry-over ContaminationContamination

Omnigrid Accent

•Complete control over the wash steps (time)

•Cycles through the wash protocol•Wash

•Sonication station

•Vacuum dry

Minimizing Carry-over Minimizing Carry-over ContaminationContamination

Microgrid II

• 2 recirculating wash baths – can be stationary (etoh)

• Main Wash station “flood and flush”

• vacuum dry

Complete control over the wash steps (time)Cycles through the wash protocol

Minimizing Carry-over Minimizing Carry-over ContaminationContamination

• 2 systems have different wash setups • BOTH were shown to be equally effective for

cleaning pins.

• Empirically determine proper wash protocol:

Protein microarray Experiment –Protein microarray Experiment –3D substrate3D substrate

• Print 5mg/ml of rabbit IgG (Invitrogen)

• 3X3 array onto the pads of a 8 Pad FAST slide (Whatman)

• Solution phase binder - 100ug/ml of FluoroLink Cy3-labeled goat anti-rabbit IgG (GE Healthcare)

Genetic Engineering News – Assay Tutorial – Jan. 2006

Binding AssayBinding Assay

• Genomic Solutions HybStation

• Binding • Washing

Protein microarray Experiment –Protein microarray Experiment –3D substrate3D substrate

Protein MicroarraysProtein Microarrays

• Traditional DNA chemistry for protein applications: Epoxy, aldehyde

• Slide E, AL (Schott Nexterion)

• Multiplexed Assay: Microarray assembled in order to assay a large number (16, 96, 384) of samples in parallel to decrease cost and increase assay reproducibility.

Multiplexed Protein Multiplexed Protein MicroarraysMicroarrays

Slide MPX

Plate MTP

Plate can be printed separately or inside a tray

Maximum printing area is available (up to 2000 probes/well)

Analysis of 96 targets simultaneously

Design conforms to the SBS standards and suited for high throughput robotic handling

Designed to eliminate cross-contamination

Chemistries optimized for DNA and protein applications

Nexterion MTP has a Unique Nexterion MTP has a Unique DesignDesign

Ultra hydrophobic pattern ensures negligible cross-contamination across wells of multiplexed substrates

2082 26971167

7331 8575 5483

532 592348

21

2

1

10

100

1000

10000

100000

Gene 51 Gene1765

Gene1833

Gene 51 Gene1765

Gene1833

Slides/Conditions

To

tal M

ed

ian

Sig

na

l

Target No Target

non-MPX MPX

1 2

3 4

5 6

7 8

1 2

3 4

5 6

7 8

Partitioned

1 2

3 4

5 6

7 8

1 2

3 4

5 6

7 8

1 2

3 4

5 6

7 8

1 2

3 4

5 6

7 8

Target added to wells 1, 4, 5, and 8 only

Simple target Complex TargetNon-partitioned PartitionedNon-partitioned

<3% contamination in partitioned wells

Contamination not detectable in partitioned wells

100

50

75

25

0

Cy3 Cy5

Nor

mal

ized

sig

nal

Afte

r hy

brid

izat

ion

Target volume: 60 µl 45 µl 30 µl Target amount: 7.5 pmol 7.5 pmol 7.5 pmol

166 nM

125 nM

250 nM

Different volumes can be used for hybridization Increased sensitivity can be achieved using lower volumes

Increased Sensitivity on Multiplexed Increased Sensitivity on Multiplexed SubstratesSubstrates

Protein Array printing on MTP Protein Array printing on MTP platesplates

• In addition to good spot morphology

• No carry over between samples

• Axis Stability - Stable positioning into EACH well of a multiwell plate to consistently position array in relation to well wall: Microgrid II, Omnigrid Accent

• Easy to use software

Microgrid Plate printingMicrogrid Plate printing

Easy to use plate printing interface: PlateArray

Wizard

Multiplex Plate Arraying on MicroGrid IIMultiplex Plate Arraying on MicroGrid II

• Capacity– 24 source plates– 16 target plates

• Source plate cooling• Plate & lid handling for

sample plates• Humidity controlled• HEPA filtered• “Soft-touch” software• Re-circulating wash baths

& high pressure pin drying• Barcode reading option

Omnigrid Accent plate printing

• Software upgrade:

Medium throughput: 6 target plates, 3 source plates at a time. Humidity, plate cooling

Multiplex Protein MicroArrayMultiplex Protein MicroArray

• Substrate: MTP 96 (Slide E or AL)

• Printer: Micrigrid II/Omnigrid Accent

• Imaging: Alpha Innotech - Novaray

• Alpha Innotech’s NovaRay ® Alpha Innotech’s NovaRay ® brings the multiplexing of brings the multiplexing of arrays to high throughput arrays to high throughput screening.screening.

• Multi-format testing capabilities– Microplates– Slides– Custom formats

• Multispectral imaging– Broad-band light source– 8 emission wavelengths

NovaRay ®: A Novel High NovaRay ®: A Novel High Throughput Multiplexed Array Throughput Multiplexed Array Detection PlatformDetection Platform

Gene Arrays

Protein Arrays

Cell Arrays

Chemical Compound Arrays

Primary Focus

Secondary Focus

NovaRay ® Supports Multiple ApplicationsNovaRay ® Supports Multiple Applications

Compatible dyes Excitation Wavelength Emission Wavelength

Cy3, Cy3.5, Alexa 546, TRITC, JOE, ROX, TAMRA 480-552 572-640

Cy3 527-552 570-620

Cy5, Cy5.5, Alexa 647, Alexa 680, Bodipy 630/650 572-640 664-750

Cy5 620-650 665-705

FITC, Alexa 488, FAM, PicoGreen, GFP 455-495 518-568

Texas Red 540-597 615-665

NIR 675-725 730-830

Phycoerythrin 480-552 664-750

NovaRay ® Supported WavelengthsNovaRay ® Supported Wavelengths

1

3

5

7

Similar images obtained in different wells on a multiplexed substrate

2

3

6

4

8

Average Log Intensity (ALI) = [Log10(Cy3+Cy5)/2]

Data from individual wells can be quantitativelycompared using regression analysis

0.5

1.0

1.5

2.0

2.5

1.0 1.5 2.0 2.5

R2=0.98

0.5

1.0

1.5

2.0

2.5

1.0 1.5 2.0 2.5ALI (Subarray 3)

ALI

(Sub

arra

y5)

R2=0.98

Average R2 = 0.96 ± 0.03

1 2 3 4 5 6 7 81 0.96 0.93 0.93 0.91 0.93 0.91 0.932 0.96 0.95 0.95 0.93 0.95 0.94 0.953 0.93 0.95 0.98 0.98 0.98 0.98 0.994 0.93 0.95 0.98 0.98 0.98 0.98 0.995 0.91 0.93 0.98 0.98 0.97 0.99 0.986 0.93 0.95 0.98 0.98 0.97 0.98 0.987 0.91 0.94 0.98 0.98 0.99 0.98 0.988 0.93 0.95 0.99 0.99 0.98 0.98 0.98

1 2 3 4 5 6 7 81 0.96 0.93 0.93 0.91 0.93 0.91 0.932 0.96 0.95 0.95 0.93 0.95 0.94 0.953 0.93 0.95 0.98 0.98 0.98 0.98 0.994 0.93 0.95 0.98 0.98 0.98 0.98 0.995 0.91 0.93 0.98 0.98 0.97 0.99 0.986 0.93 0.95 0.98 0.98 0.97 0.98 0.987 0.91 0.94 0.98 0.98 0.99 0.98 0.988 0.93 0.95 0.99 0.99 0.98 0.98 0.98

Subarray

A pair-wise comparison can be used to assessoverall reproducibility

Excellent Reproducibility (Excellent Reproducibility (R2> 0.9 ) on Multiplexed ) on Multiplexed SubstratesSubstrates

Signal vs Exposure Time

R2 = 0.9938

0

10000

20000

30000

40000

50000

60000

500 ms 1000 ms 2000 ms 3000 ms 4000 ms 5000 ms

Expsoure time (milliseconds)

Rel

ativ

e In

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sity

Val

ue

• Multispot array target.

• Linear imaging results with increase in exposure time.

•High correlation between increasing exposure time (R2 = 0.9938)

Linear Response: User ValidationLinear Response: User Validation

Integrated Muliplex PlatformIntegrated Muliplex Platform

Results !Results !

• Cell based time-point studies

• Lead optimization• Clinical trials• Biomarker screening and

validation• Cell Based Assays• Enzyme Assays • Functional Assays • In Vitro Toxicology • Biomarker screening and

validation• Antiviral Assays• Novel Targets Panel

Multiplex Array ApplicationsMultiplex Array Applications

Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray

• Print 0.1 mg/ml rabbit IgG onto Nexterion Slide E MTP 96 Epoxy substrate • 3X3 subarray in each well – Microgrid II

• Solution phase binder - 10ug/ml of FluoroLink Cy3-labeled goat anti-rabbit IgG (GE Healthcare) (Schott Nexterion protocol)

• Scan image with the Alpha Innotech Novaray

Results !Results !

Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray

Cy3 labeled

anti-rabbit IgG bound to rabbit

IgG

Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray

Anti-rabbit IgG Negative

Anti-IgG

Anti-HSA

Probes detected

All IgG HSA

IgG and HSA proteins are specifically detected by their respective antibodies

MPX

MTP

No carry over

Anti-STP

Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray

SummarySummary

• SCHOTT: MPX slide and plate formats

• Simultaneous biological sample analysis• Versatile assay design possibilities• Critical for diagnostic/applied research

• Genomic Solutions: MicroGrid II & OmniGrid Accent

• OmniGrid Accent medium throughput plate arraying• MicroGrid II high throughput• Available to new & existing customers

• Alpha Innotech: NovaRay® Plate reader• Supports multiple wavelength assays• High resolution capability• Multi-chamber slide and 96 well plate imaging

Protein Microarray WorkflowProtein Microarray Workflow

AcknowledgementsAcknowledgements

• Genomic Solutions

– Dr. Jeremy Clarke

– Dr. Sally Johnston

– Dr. Jennifer Owen

– Dr. Jim Galt

– Dr. Hamid Khoja

– Judy Thompson

• Schott Nexterion

– Dr. Rajendra Redkar

– Dr. Luis Burzio

– Heather Jakes

• Alpha Innotech

Dr. Mike Collier