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
HydroGelTM Coated Slides
HydroGelTM
Performance Validation
• Printing compatibility• Inherent fluorescent background• Loading capacity of substrate• Protein compatibility• Nonspecific background• Multiplexed assay performance
Printing Compatibility
Packard BioChip ArrayerTM (Piezo)
Feature Size ~200 um
Packard SpotArray 24 (Split Pin)
Feature Size ~150 um
HydroGelTM 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 BackgroundTexas Red CY3 CY5 FITC
1.62 X2.44 X 2.91 X4.39 X
1.79 X 1.70 X1.76 X 4.14 X
190 X498 X 528 X 34 X
HydroGelTM coated slide
Aldehyde glass
Nitrocellulose coated slide
Blank substrates were scanned on a ScanArrayTM 5000 microarray scanner in the channels indicated (laser power:100%, PMT gain: 75%).
Protein Loading Capacity
Immobilization of IgG
0
20
40
60
80
100
120
140
160
0.5 1 2 4 8
Printing Concentration (mg/mL)
fmol
es p
er m
m2
HydroGel
Aldehyde Glass
Immobilization of Streptavidin
0
50
100
150
200
250
300
350
0.5 1 2 4 8
Printing Concentration (mg/mL)
fmol
es p
er m
m2 HydroGel
Aldehyde Glass
IgG and Streptavidin were printed on HydroGelTM 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
startingending
~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
0
1
2
3
4
5
6
7
8
9
HydroGel slide Nitrocellulose slide Aldehyde glass
Sig
nal
/Bac
kgro
un
d
Anti-bovine IgG
Anti-avidin
HydroGelTM coated slide
aldehyde glassnitrocellulose
coated slide
(negative control)
Low Nonspecific Background
Images courtesy of Dr. Brian Haab (Van Andel Research Institute, Grand Rapids, MI).
Poly-lysine based slide HydroGelTM Coated Slide
Targets: Cy3- and Cy5-labeled patient serum samples
Protein Compatibility
Alkaline Phosphatase Response
Enzyme printing concentration (ng/ml)
0.01 0.1 1
Net
fluo
resc
ence
(rf
u)
1e+2
1e+3
1e+4
1e+5
HydroGel
Nitrocellulose slide
Minimal Detectable Limits
Predicted(ng/ml)*
Observed(ng/ml)
HydroGel™ CoatedSlide
0.015 0.019
Nitrocellulose Slide 0.028 0.076**
* 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 10-1000 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 HydroGelTM 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 chipA: Cell culture media as negative control (left) showing low non-specific bindingB: Conditioned media (center) indicating cells produced IL-8, GCSF and IL –6C: Cell lysates (right) containing IL-1b, GCSF and IL-8 but lacking IL-6
A B C
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-6
IL-1b
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
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