microporous nitrocellulose surfaces for increased ... · nova™ – 10 times higher signal-to-...

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

Protein Microarrays

Infrared / University of Nottingham

Fluorescent / OEM Partner

Colorometric / Iwate Medical University

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

Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide

Porous Nitrocellulose Film

Scanning Electron Micrograph Representation of Planar Surface

Porous Nitrocellulose Film

Protein Spotting on Surface

Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide

Porous Nitrocellulose Film

Protein Spotting on Surface

Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide

Porous Nitrocellulose Film

Protein Spotting on Surface Protein Deposition Through Matrix

Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide

Porous Nitrocellulose Film

Protein Spotting on Surface Protein Deposition Through Matrix

Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide

Optimized Porosity

Porous Nitrocellulose Film

Protein Spotting on Surface Protein Deposition Through Matrix

Grace BioLabs 16-Pad Film Slide Conventional 2-D Glass Slide

Optimized Porosity

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

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

Porous Nitrocellulose Film Slides

AVID™ NOVA™ SuperNOVA™

Optimized For Different Protein Microarray Applications

Porous Nitrocellulose Film Slides

For Assays That Require the

Highest Binding Capacity

AVID™

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

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

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

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

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

Porous Nitrocellulose Film Slides

For Detection Requiring the

Lowest Fluorescent Background

NOVA™

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

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

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

Porous Nitrocellulose Film Slides

For High Capacity, Fluorescence-

Based Protein Arrays

SuperNOVA™

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

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

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

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

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

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)

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

ProPlate® Chambers

Improve Assay Kinetics

Minimize Sample Use

Simplify Workflow

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

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

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

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