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Microporous Nitrocellulose Surfaces For
Increased Sensitivity and Dynamic Range of
Protein Microarrays
Porous Nitrocellulose Film Slides
Fluidic Seals and Chambers
Extract the most out of your experiment:
Maximize Signal and Quality
Minimize Variation
Minimize Sample Use
Improve Workflow
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Protein Microarrays
Infrared / University of Nottingham
Fluorescent / OEM Partner
Colorometric / Iwate Medical University
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Porous Nitrocellulose Film Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
3-Dimensional Matrix Increased Surface Area = Increased Binding Capacity
Non-Covalent Attachment Chemistry Enhances Retention of Proteins’ 3-D Conformation
Enhances Retention of Biological Activity
Key Advantages of Porous Nitrocellulose
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Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
Porous Nitrocellulose Film
Scanning Electron Micrograph Representation of Planar Surface
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Porous Nitrocellulose Film
Protein Spotting on Surface
Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
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Porous Nitrocellulose Film
Protein Spotting on Surface
Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
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Porous Nitrocellulose Film
Protein Spotting on Surface Protein Deposition Through Matrix
Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
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Porous Nitrocellulose Film
Protein Spotting on Surface Protein Deposition Through Matrix
Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
Optimized Porosity
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Porous Nitrocellulose Film
Protein Spotting on Surface Protein Deposition Through Matrix
Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
Optimized Porosity
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Porous Nitrocellulose Film
Protein Spotting on Surface Protein Deposition Through Matrix
Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide
3-Dimensional Matrix • Maximizes Number of Weak Bonding Forces
• Enhances:
Retention of Protein
Retention of 3-D Conformation
Retention of Biologic Activity
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What Parameters Impact
Variability In Your Data?
Substrates
Assay Conditions
Data Collection
Microarray Printing
Sample Preparation
Data Analysis
Microarray Pre-Treatment
Post-Assay Treatment
Substrates
Assay Conditions
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Porous Nitrocellulose Film Slides
AVID™ NOVA™ SuperNOVA™
Optimized For Different Protein Microarray Applications
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Porous Nitrocellulose Film Slides
For Assays That Require the
Highest Binding Capacity
AVID™
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AVID™ – 500 Times the Binding
Capacity of 2-D Surfaces
0
10
20
30
40
50
60
AVID Gentel PATH Aminosilane
Bo
un
d Ig
G (
µg
/cm
2)
• N = 4 film slides per slide type, 20 technical replicates per slide
• Data are derived from normalized, background-subtracted fluorescence collected at 532 nm from spotted goat IgG-Cy3
0
0.1
0.2
AVID Gentel PATH Aminosilane
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AVID™ – 7 Orders of
Magnitude in Protein Binding
1
10
100
1000
10000
100000
1000000
10000000
100000000
0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000
Spotted IgG (pg)
RF
U
6 Orders of Magnitude
Linearity
(R2 = 0.999)
• N = 4 film slides per slide type, 20 technical replicates per IgG concentration per slide
• Data are normalized, background-subtracted fluorescence collected at 532 nm from spotted goat IgG-Cy3
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AVID™ – Dynamic Range Of Protein
Binding Versus 2-D Surfaces
0.001
0.01
0.1
1
10
100
1000
10000
100000
1000000
10000000
100000000
0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000
Spotted IgG (pg)
RF
U
4 - 5 Orders of Magnitude
Linearity
(R2 = 0.999)
Gentel PATH
Aminosilane
AVID 6 Orders of Magnitude
Linearity
(R2 = 0.999)
Truncated Dynamic Range
• N = 4 film slides per slide type, 20 technical replicates per IgG concentration per slide
• Data are normalized, background-subtracted fluorescence collected at 532 nm from spotted goat IgG-Cy3
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AVID™ – 2 to 7 Times the Binding
Capacity Than Competing Films
0
10
20
30
40
50
60
AVID Whatman FAST Schott NC-C Schott NC-N
Bo
un
d Ig
G (
µg
/cm
2)
• N = 4 film slides per slide type, 20 technical replicates per slide
• Data are derived from normalized, background-subtracted fluorescence collected at 532 nm from spotted goat IgG-Cy3
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AVID™ – Higher Signal in
Downstream Microarray Assays
0
5
10
15
20
25
AVID Schott NC-N
RU
0
1000
2000
3000
4000
5000
AVID Schott NC-N
RF
U
Colorimetric Fluorescence
• N = 4 film slides per slide type, 20 technical replicates per slide
• All data obtained from spotting concentrations within the linear dynamic range of detection methods employed
• Colorimetric data are normalized and background-subtracted, collected after HRP/AEC staining from spotted goat IgG assayed
with anti-goat IgG
• Fluorescence data are normalized and background-subtracted, collected at 532 nm from spotted rabbit serum assayed with goat
anti-rabbit IgG
4x Signal 2.5x Signal
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Porous Nitrocellulose Film Slides
For Detection Requiring the
Lowest Fluorescent Background
NOVA™
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0.1
1
10
100
1000
10000
100000
1000000
10000000
100000000
0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000
Spotted IgG (pg)
RF
U
6 Orders of Magnitude
Linearity
(R2 = 0.998)
• N = 4 film slides per slide type, 20 technical replicates per IgG concentration per slide
• Data are normalized, background-subtracted fluorescence collected at 532 nm from spotted goat IgG-Cy3
NOVA™ – 7 Orders of Magnitude
in Protein Binding
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NOVA™ – Low Autofluorescence
Compared to Competing Porous Films
0
5000
10000
15000
20000
25000
30000
NOVA Schott NC-N Schott NC-C FAST
RF
U
532 nm
635 nm
7 x
20 x
40 x
< 700
• N = 14 film slides for NOVA, N = 8 for all other film slides
• 532 nm data obtained at 33% laser power, 500 PMT; 635 nm data obtained at 100% laser power, 650 PMT
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NOVA™ – 10 Times Higher Signal-to-
Noise Than Competing Film Slides
0
100
200
300
0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000
Serum Protein (pg)
Sig
nal-
to-N
ois
e
• Normal rabbit serum spotted, blocked, and assay with goat anti-rabbit IgG
• N = 4 film slides per slide type, 20 technical replicates per slide
• Data are derived from fluorescence intensities collected at 532 nm
Whatman FAST
Schott NC-N
Schott NC-C
NOVA Up to 10x Higher Signal-to-Noise
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Porous Nitrocellulose Film Slides
For High Capacity, Fluorescence-
Based Protein Arrays
SuperNOVA™
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SuperNOVA™ – Protein Binding
Capacity of AVID
0
10
20
30
40
50
60
SuperNOVA AVID Whatman
FAST
Schott NC-C Schott NC-N
Bo
un
d Ig
G (
µg
/cm
2)
2 - 7x Binding Capacity
• N = 4 film slides per slide type, 20 technical replicates per slide
• Data are derived from normalized, background-subtracted fluorescence collected at 532 nm from spotted goat IgG-Cy3
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SuperNOVA ™ – 7 Orders of
Magnitude in Protein Binding
1
10
100
1000
10000
100000
1000000
10000000
100000000
0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000
Spotted IgG (pg)
RF
U
6 Orders of Magnitude
Linearity
(R2 = 0.9999)
• N = 4 film slides per slide type, 20 technical replicates per IgG concentration per slide
• Data are normalized, background-subtracted fluorescence collected at 635 nm from spotted goat IgG-Cy5
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SuperNOVA™ – Low Autofluorescence
of NOVA
0
5000
10000
15000
20000
25000
30000
SuperNOVA NOVA Schott NC-N Schott NC-C FAST
RF
U
532 nm
635 nm
< 400
10 x
30 x
60 x
• N = 14 film slides for SuperNOVA and NOVA, N = 8 for all other film slides
• 532 nm data obtained at 33% laser power, 500 PMT; 635 nm data obtained at 100% laser power, 650 PMT
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SuperNOVA ™ - Excellent Signal-to-Noise
SuperNOVA™
300 µm
250 µm
160 µm
• Spotting Concentration 5 ug/ml IgG
• 532 nm data obtained at 33% laser power, 600 PMT
• GenePix Brightness / Contrast: 71 / 75
1.5
2.3
2.1
Schott NC-N
Schott NC-C
Whatman FAST
Spot Diameter
170 µm
S/N
51.3
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SuperNOVA™ – 25 Times Higher
Signal-to-Noise
0
100
200
300
400
500
0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000
Serum Protein (pg)
Sig
na
l-to
-No
ise
Whatman FAST
Schott NC-N
Schott NC-C
SuperNOVA
NOVA
• Normal rabbit serum spotted, blocked, and assay with goat anti-rabbit IgG
• N = 4 film slides per slide type, 20 technical replicates per slide
• Data are derived from fluorescence intensities collected at 532 nm
Up to 25x Higher Signal-to-Noise
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SuperNOVA – Extended Dynamic
Range in Fluorescent Assays
0.1
1
10
100
1000
10000
100000
1000000
10000000
0.0001 0.001 0.01 0.1 1 10 100 1000 10000 100000
Serum Protein (pg)
RF
U
SuperNOVA
Whatman FAST
Schott NC-N
Schott NC-C
• Normal rabbit serum spotted, blocked, and assay with goat anti-rabbit IgG
• N = 4 film slides per slide type, 20 technical replicates per slide
• Data are normalized, background-subtracted fluorescence collected at 532 nm
Gain 3 - 4 Orders of Magnitude
with SuperNOVA 6 Orders of Magnitude
Linearity
(R2 = 0.999)
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What Parameters Impact
Variability In Your Data?
Substrates
Assay Conditions
Data Collection
Microarray Printing
Sample Preparation
Data Analysis
Microarray Pre-Treatment
Post-Assay Treatment
Substrates
Assay Conditions
AVID™ – 2 - 7 Times Higher Binding Capacity
NOVA™ – 7 - 40 Times Lower Autofluorescence
SuperNOVA™ – Up to 25 Times Higher Signal-to-Noise
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ProPlate® Chambers
Improve Assay Kinetics
Minimize Sample Use
Simplify Workflow
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Reduced Variability With ProPlate®
0%
10%
20%
30%
40%
50%
Intra-Spot Intra-Slide Inter-Slide
Co
eff
icie
nt
of
Vari
ati
on
ProPlate
CoverSlip
• All data obtained from spotting concentration of 1 mg/ml (within the linear dynamic range of detection)
• Fluorescence data are normalized and background-subtracted, collected at 532 nm from spotted goat IgG assayed with rabbit anti-goat IgG-TRITC
(CoverSlip – 50 µl, 1:25,000 Dilution; 4-well ProPlate – 300 µ/well, 1:150,000 Dilution)
• Intra-Spot CV data are pixel variation per spot, N = 64 spot replicates (over 4 microarrays)
• Intra-Slide CV data are spot variation per array, N = 4 microarrays (mean of N = 16 spot replicates/array)
ProPlate
Cover Slip
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Substrates
What Parameters Impact
Variability In Your Data? Assay Conditions
Data Collection
Microarray Printing
Sample Preparation
Data Analysis
Microarray Pre-Treatment
Post-Assay Treatment
Assay Conditions
ProPlate® Chambers – Improved Assay Kinetics
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Substrates
Assay Conditions
Data Collection
Microarray Printing
Sample Preparation
Data Analysis
Microarray Pre-Treatment
Post-Assay Treatment
Substrates
Assay Conditions
AVID™ – 2 - 7 Times Higher Binding Capacity
NOVA™ – 7 - 40 Times Lower Autofluorescence
SuperNOVA™ – Up to 25 Times Higher Signal-to-Noise
ProPlate® Chambers – Improved Assay Kinetics
Grace Bio-Labs Offers A
Simple Microarray System
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Robust Microarray Platform ONCYTE® Porous Nitrocellulose
Film Slides provide: Highest protein binding capacity
Lowest fluorescent backgrounds
Highest signal-to-noise
ProPlate® Chambers provide: Increased assay kinetics
Decreased assay variability
For Increased Sensitivity and Dynamic
Range of Protein Microarrays