BioChip Ventures Division a 3-Dimensional Microarray Substrate

What is a microarray? probe target Advantages multiplexing and miniaturization  throughput parallel analysis sample volume reduction

Protein Microarray Applications * DNA - protein interaction * Protein - protein interactions * Enzyme-substrate analysis * Protein profiling * Antibody characterization * Small molecule screening Image courtesy of Dr. Gavin MacBeath, Bauer Center for Genomics Research, Harvard University

Desirable Substrate Properties for Protein Microarray Applications Protein compatible High probe loading capacity Low inherent fluorescence and nonspecific binding background Consistent, uniform product Ease of use

HydroGel TM Coated Slides

HydroGel TM Performance Validation Printing compatibility Inherent fluorescent background Loading capacity of substrate Protein compatibility Nonspecific background Multiplexed assay performance

Printing Compatibility Packard BioChip Arrayer TM (Piezo) Feature Size ~200 um Packard SpotArray 24 (Split Pin) Feature Size ~150 um HydroGel TM coated slides are compatible with both contact and non-contact printers (examples shown above). This was verified on two other types of commercially available and “home-made” instruments.

Inherent Fluorescent Background Texas RedCY3CY5FITC 1.62 X 2.44 X 2.91 X 4.39 X 1.79 X1.70 X1.76 X 4.14 X 190 X498 X 528 X 34 X HydroGel TM coated slide Aldehyde glass Nitrocellulose coated slide Blank substrates were scanned on a ScanArray TM 5000 microarray scanner in the channels indicated (laser power:100%, PMT gain: 75%).

Protein Loading Capacity IgG and Streptavidin were printed on HydroGel TM coated slides and aldehyde glass slides at the indicated concentrations to compare loading capacity.

1.9 µm per section in Z axis Protein Penetration Demonstrated by Confocal Fluorescent Microscope Measurement starting ending ~70% penetration of a 160 kD protein

Print probes Incubate with target sample Immobilize and wash Wash and detect

Non-Specific Background in a Direct Fluorescence Assay on Serum Anti-bovine IgG Anti-avidin HydroGel TM coated slide aldehyde glass nitrocellulose coated slide (negative control)

Low Nonspecific Background Images courtesy of Dr. Brian Haab (Van Andel Research Institute, Grand Rapids, MI). Poly-lysine based slide HydroGel TM Coated Slide Targets: Cy3- and Cy5-labeled patient serum samples

Protein Compatibility * Calculated as the X value when Y is set to 2- fold the standard deviation of the background ** High inherent fluorescence of this substrate masks the signal generated by the two lowest enzyme concentrations.

ELISA: A Powerful Research Tool Representative commercial ELISA for IFN-  shows detection range of approximately pg/mL (2 log dynamic range)

Detection Complex For Sandwich Assays Capture antibody Target (cytokine) Biotinylated detection antibody Texas Red conjugated Streptavidin

Multiplexed Cytokine Assay IL-1  TNF-  IL-13 Neg. control IL-6 IFN-  IL-2 Det. Control replicates IL-1  TNF-  IL-13 Neg. control IL-6 IFN-  IL-2 Det. Control replicates Tissue culture media + 10% FBS only Spiked with 158 pg/mL of each cytokine

Standard Curve for TNF-  TNF- 

Regression Analysis of Cytokine Assays Trimmed standard curves for six cytokines B. TNF-  C. IL-1  D. IL-2 F. IL-13 G. IFN-  E. IL-6

Substrate Comparison A.B.C. Nitrocellulose slide Aldehyde glass Comparison of standard curves derived for IL-6 in multiplexed assays on HydroGel TM coated slides, nitrocellulose coated slides and aldehyde-derivatized glass slides. HydroGel Nitrocellulose slide Aldehyde glass slide

43 Cytokine Antibody Chip Each probe is printed in quadruplicate (350 pL/spot) at 500 um spacing.

Qualitative Screening Human ER-negative breast cancer cells MDA-MB-231 were screened with a 43 cytokine antibody chip A: Cell culture media as negative control (left) showing low non-specific binding B: Conditioned media (center) indicating cells produced IL-8, GCSF and IL –6 C: Cell lysates (right) containing IL-1b, GCSF and IL-8 but lacking IL-6 A BC IL-8 IL-6 GCSF Control IL-1b Biotin-IgG

Ratiometric results Results indicate that MB231 cells secret IL-6 while IL-1b retained inside of cells –Top images are the overlapping of cell culture supernatant (red) and cell lysate (green) –Bottom is the ratio correlation map between red and green IL-6 IL-1b

Summary compatible with both contact and non-contact printing low inherent fluorescence and nonspecific background higher functional protein loading capacity 3-dimensional, hydrophilic environment seems to maintain protein structure and promotes functionality superior assay performance high sensitivity broad dynamic range