Download Microarray Technology

a 3-Dimensional Microarray Substrate
BioChip Ventures Division
What is a microarray?
Advantages
• multiplexing and miniaturization
•  throughput
• parallel analysis
target
• sample volume reduction
probe
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
TM
HydroGel
•
•
•
•
•
•
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 Background
HydroGelTM
coated slide
Texas Red
CY3
CY5
FITC
1.76 X
4.14 X
1.79 X
1.70 X
2.44 X
4.39 X
2.91 X
1.62 X
498 X
528 X
34 X
190 X
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 Streptavidin
350
160
140
HydroGel
120
100
80
60
fmoles per mm2
fmoles per mm2
Immobilization of IgG
Aldehyde Glass
40
20
0
300
HydroGel
250
Aldehyde Glass
200
150
100
50
0
0.5
1
2
4
Printing Concentration (mg/mL)
8
0.5
1
2
4
8
Printing Concentration (mg/mL)
IgG and Streptavidin were printed on HydroGelTM coated slides and
aldehyde glass slides at the indicated concentrations to compare loading
capacity.
Protein Penetration Demonstrated by Confocal
Fluorescent Microscope Measurement
~70% penetration of a
160 kD protein
starting
ending
1.9 µm per section in Z axis
Print probes
Immobilize and wash
Incubate with target
sample
Wash and detect
Non-Specific Background in a Direct
Fluorescence Assay on Serum
Anti-bovine IgG
Anti-avidin
(negative control)
HydroGelTM
coated slide
nitrocellulose
coated slide
9
Signal/Background
8
7
6
5
4
3
2
1
0
HydroGel slide
Nitrocellulose slide
Aldehyde glass
aldehyde glass
Low Nonspecific Background
Poly-lysine based slide
HydroGelTM Coated
Slide
Targets: Cy3- and
Cy5-labeled patient
serum samples
Images courtesy of Dr.
Brian Haab (Van Andel
Research Institute,
Grand Rapids, MI).
Protein Compatibility
Alkaline Phosphatase Response
Minimal Detectable Limits
1e+5
Predicted
(ng/ml)*
Observed
(ng/ml)
HydroGel™ Coated
Slide
0.015
0.019
Nitrocellulose Slide
0.028
0.076**
Net fluorescence (rfu)
HydroGel
1e+4
Nitrocellulose slide
1e+3
* Calculated as the X value when Y is set to 2fold the standard deviation of the background
1e+2
0.01
0.1
Enzyme printing concentration (ng/ml)
1
** 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-g shows detection range of
approximately 10-1000 pg/mL (2 log dynamic range)
Detection Complex For Sandwich Assays
Texas Red conjugated Streptavidin
Biotinylated detection antibody
Target (cytokine)
Capture antibody
Multiplexed Cytokine Assay
Tissue culture media + 10% FBS only
IL-1b
IL-6
TNF-a
IFN-g
IL-13
IL-2
Neg. control
Det. Control
replicates
Spiked with 158 pg/mL of each cytokine
IL-1b
IL-6
TNF-a
IFN-g
IL-13
IL-2
Neg. control
Det. Control
replicates
Standard Curve for TNF-a
TNF-a
Regression Analysis of Cytokine Assays
B. TNF-a
C. IL-1b
D. IL-2
E. IL-6
F. IL-13
G. IFN-g
Trimmed standard curves for six cytokines
Substrate Comparison
HydroGel
A.
Nitrocellulose slideslide
Nitrocellulose
B.
Aldehyde glass
Aldehyde
glass slide
C.
Comparison of standard curves derived for IL-6 in multiplexed assays on HydroGelTM
coated slides, nitrocellulose coated slides and aldehyde-derivatized glass slides.
43 Cytokine Antibody Chip
Each probe is printed in quadruplicate
(350 pL/spot) at 500 um spacing.
Qualitative Screening
A
B
C
Biotin-IgG
IL-1b
IL-8
IL-6
Control
GCSF
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
Ratiometric results
IL-6
IL-1b
IL-6
IL-1b
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
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