Overview of SCHOTT’s protein micro-array slide surfaces
1
Binding capacity and signal intensity. (0,25) Signal/ Background ratio (0,25) Probe target linearity (0,2) Sensitivity (0,15) Print buffer tolerance (0,15) Printed slide storage conditions Slide H +++ ++++ +++ ++++ ++ -20 °C Slide P +++ ++++ +++ ++++ ++ -20 °C Slide NC ++++ ++ ++++ ++ ++++ +20 °C Slide E +++ +++ +++ +++ +++ -20 °C Slide AL ++ ++ ++ ++ ++ -20 °C SCHOTT’s Protein Microarray Slides: Choosing the right coating Dietmar Knoll, Corinna Schwung, Alistair Rees and Rüdiger Dietrich SCHOTT Technical Glass Solutions GmbH., Otto-Schott-Strasse 13, 07745 Jena, Germany +49 3641 681 4066 NEXTERION ® protein microarray surfaces MTP-96 well SBS microplate format 16-pad slide Standard 25 x 75 mm slide Customized 8-well slide (customized) 2-pad slide SCHOTT Nexterion The lower number of protein species to be printed allows smaller array sizes. This allows lower assay volumes and finally cost/time savings due to multiplexing Substrate Formats Conventional 2D surfaces (<10nm) Metallic surfaces 3-D surfaces Epoxy, Aldehyde-silane Gold coatings Protein Microarray Substrates 3-D thick film coatings (0.1–200µm) 3-D thin film coatings (10–100nm) Nitrocellulose, Agarose coatings Polymer and hydrogel type Conventional 2-D Substrates CH 2 CH O l CH 2 CH O l CH 2 CH O l epoxy groups Immobilization •• O NH 2 OH NH P P Remarks 1. Robust and reproducible coatings 2. Poor stabilization of protein structure 3. Storage of printed arrays at –20 °C Examples 1. Epoxysilane (NEXTERION ® E) 2. Aldehydesilane (NEXTERION ® AL) 3-D Thick Film Coatings- Nitrocellulose SEM image of NEXTERION ® Slide NC Schott Nexterion® Slide NC Diagram showing increased capacity due to pore structure Examples 1. NC (NEXTERION ® Slide NC-N and -C) 2. Agarose Remarks 1. Nitrocellulose is widely used surface for proteins 2. High print buffer tolerance (e.g. lysate detergents) 3. Stabilization of protein structure (long shelf life) 4. Can have high background 5. Opaque NC coating not compatible some detection methods 3-D Thin Film Coatings Cross links Reactive group Polymer - selected to provide lowest unspecific binding and to stabilize protein structure Examples 1. Thin film coatings (NEXTERION ® H) 2. Polymer coatings (NEXTERION ® P) Remarks 1. Very low non-specific binding 2. Very high S/N ratios 3. Stabilization of protein structure 4. Restricted choice of print buffers 5. Storage of printed arrays at – 4 or –20 °C Binding Capacity and Signal Intensities •Higher signal intensities for 3-D surfaces especially for Nitrocellulose slides •Thin film 3-D substrates have higher binding capacities than conventional 2-D substrates •Highest signal intensities for Slide NC due to higher intrinsic autoflourescence of the nitrocellulose Background •Highest background for Slide NC •Very low unspecific binding for Slide P and Slide H •Low background signals for conventional 2 D substrates Signal / Background ratio Benchmark Test Results SCHOTT NEXTERION ® has been supplying the microarray slide market for nearly 10 years, and is committed providing the best substrates SCHOTT NEXTERION ® provides a broad range of high quality coated glass substrates suitable for protein microarrays Summary Printed probe: rabbit anti goat Ab (1,2mg/ml) – target: IgG Goat •Highest S/B ratios for Slide P and Slide H •S/B ratios of Nitrocellulose slides and conventional 2D slides are comparable •Excellent correlation between probe and target concentration and signal intensities on all slides Data have been normalized to allow data comparison Printed probe: rabbit anti goat Ab – target: IgG Goat (3,125µg/ml) signal intensity colours refer to antibody concentration printed Slide H Slide P Slide E Slide AL Slide NC 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1000000 02 RaG 0,01mg/ml 3125 03 RaG 0,025mg/ml 3125 04 RaG 0,1mg/ml 3125 05 RaG 0,3mg/ml 3125 06 RaG 0,6mg/ml 3125 07 RaG 1,2mg/ml 3125 Slide H Slide P Slide E Slide AL Slide NC signal intensity 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 02 RaG 0,01mg/ml 3125 03 RaG 0,025mg/ml 3125 04 RaG 0,1mg/ml 3125 05 RaG 0,3mg/ml 3125 06 RaG 0,6mg/ml 3125 07 RaG 1,2mg/ml 3125 colours refer to antibody concentration printed 0 Slide H Slide P Slide E Slide AL Slide NC signal intensity colours refer to target concentration (IgG goat) 0 100 200 300 400 500 600 3125 625 125 25 5 1 signal intensity colours refer to target concentration (IgG goat) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 intensity 3125 625 125 25 5 1 Slide H Slide P Slide E Slide AL Slide NC Slide H Slide P Slide E Slide AL Slide NC Printed probe: anti osteopontin Ab – target: Osteopontin S/B ratio Printed protein concentration in mg/ml colours refer to target con- centration (osteopontin) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 3125 ng/ml 625 ng/ml 125 ng/ml 25 ng/ml 5 ng/ml 1 ng/ml Printed protein concentration in mg/ml Slide H Slide P Slide E Slide AL Slide NC 0 200 400 600 800 1000 1200 1400 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 0,03125 0,0625 0,125 0,25 0,5 1 3125 ng/m l 625 ng/m l 125 ng/m l 25 ng/m l 5 ng/m l 1 ng/m l colours referto targetcon- centration(osteopontin) S/B ratio
-
Upload
schott -
Category
Technology
-
view
1.306 -
download
2
description
Choosing the best slide coating chemistry for your protein micro-array application. Poster presented at the Advances in Microarray Technology 2008 conference in Barcelona, Spain.
Transcript of Overview of SCHOTT’s protein micro-array slide surfaces
Binding capacity and signal
intensity. (0,25)
Signal/Background ratio
(0,25)Probe target linearity (0,2)
Sensitivity (0,15)
Print buffer tolerance (0,15)
Printed slide storage conditions
Slide H
+++
++++
+++
++++
++
-20 °C
Slide P
+++
++++
+++
++++
++
-20 °C
Slide NC
++++
++
++++
++
++++
+20 °C
Slide E
+++
+++
+++
+++
+++
-20 °C
Slide AL
++
++
++
++
++
-20 °C
SCHOTT’s Protein Microarray Slides: Choosing the right coatingDietmar Knoll, Corinna Schwung, Alistair Rees and Rüdiger Dietrich
SCHOTT Technical Glass Solutions GmbH., Otto-Schott-Strasse 13, 07745 Jena, Germany +49 3641 681 4066
NEXTERION® protein microarray surfaces
MTP-96 well SBS microplate format
16-pad slide
Standard 25 x 75 mm slide Customized
8-well slide (customized)2-pad slide
SCHOTT Nexterion
The lower number of protein species to be printed allows smaller array sizes.
This allows lower assay volumes and finally cost/time savings due to multiplexing.
Substrate Formats
Conventional 2D surfaces (<10nm) Metallic surfaces 3-D surfaces
Epoxy, Aldehyde-silane Gold coatings
Protein Microarray Substrates
3-D thick film coatings (0.1–200µm) 3-D thin film coatings (10–100nm)
Nitrocellulose, Agarose coatings Polymer and hydrogel type
Conventional 2-D Substrates
CH2
CHO l
CH2
CHO l
CH2
CHO l
epoxy groups
Immobilization••
O
NH2
OH
NH
P
P
Remarks
1. Robust and reproducible coatings
2. Poor stabilization of protein structure
3. Storage of printed arrays at –20 °C
Examples
1. Epoxysilane (NEXTERION® E)
2. Aldehydesilane (NEXTERION® AL)
3-D Thick Film Coatings-Nitrocellulose
SEM image of NEXTERION® Slide NC
Schott Nexterion® Slide NCDiagram showing increased capacity due to pore structure
Examples
1. NC (NEXTERION® Slide
NC-N and -C)
2. Agarose
Remarks
1. Nitrocellulose is widely used surface for proteins
2. High print buffer tolerance (e.g. lysate detergents)
3. Stabilization of protein structure (long shelf life)
4. Can have high background
5. Opaque NC coating not compatible some
detection methods
3-D Thin Film Coatings
Cross links
Reactive groupPolymer - selected to provide lowest unspecific
binding and to stabilize protein structure
Examples
1. Thin film coatings (NEXTERION® H)
2. Polymer coatings (NEXTERION® P)
Remarks
1. Very low non-specific binding
2. Very high S/N ratios
3. Stabilization of protein structure
4. Restricted choice of print buffers
5. Storage of printed arrays at – 4 or –20 °C
Binding Capacity and Signal Intensities
• Higher signal intensities for 3-D surfaces
especially for Nitrocellulose slides
• Thin film 3-D substrates have higher
binding capacities than conventional 2-D
substrates
• Highest signal intensities for Slide NC
due to higher intrinsic autoflourescence
of the nitrocellulose
Background
• Highest background for Slide NC
• Very low unspecific binding for
Slide P and Slide H
• Low background signals for
conventional 2 D substrates
Signal / Background ratio
Benchmark Test Results
SCHOTT NEXTERION® has been supplying the
microarray slide market for nearly 10 years, and is
committed providing the best substrates
SCHOTT NEXTERION ® provides a broad range of
high quality coated glass substrates suitable for
protein microarrays
Summary
Printed probe: rabbit anti goat Ab (1,2mg/ml) – target: IgG Goat
• Highest S/B ratios for Slide P
and Slide H
• S/B ratios of Nitrocellulose slides
and conventional 2D slides are
comparable
• Excellent correlation between
probe and target concentration
and signal intensities on all slides
Data have been normalized to allow data comparison
Printed probe: rabbit anti goat Ab – target: IgG Goat (3,125µg/ml)
sign
al in
tens
ity
colours refer to antibody concentration printed
Slide H Slide P Slide E Slide AL Slide NC
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
02 RaG 0,01mg/ml 3125
03 RaG 0,025mg/ml 3125
04 RaG 0,1mg/ml 3125
05 RaG 0,3mg/ml 3125
06 RaG 0,6mg/ml 3125
07 RaG 1,2mg/ml 3125
Slide H Slide P Slide E Slide AL Slide NC
sign
al in
tens
ity
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
02 RaG 0,01mg/ml 3125
03 RaG 0,025mg/ml 3125
04 RaG 0,1mg/ml 3125
05 RaG 0,3mg/ml 3125
06 RaG 0,6mg/ml 3125
07 RaG 1,2mg/ml 3125
colours refer to antibody concentration printed
0
Slide H Slide P Slide E Slide AL Slide NC
sign
al in
tens
ity
colours refer to target concentration (IgG goat)
0
100
200
300
400
500
600
3125
625
125
25
5
1
sign
al in
tens
ity
colours refer to target concentration (IgG goat)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
inte
nsi
ty
3125
625
125
25
5
1
Slide H Slide P Slide E Slide AL Slide NC
Slide H Slide P Slide E Slide AL Slide NC
Printed probe: anti osteopontin Ab – target: Osteopontin
S/B
rat
io
Printed protein concentration in mg/ml
colours refer to target con-centration (osteopontin)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0,03
125
0,06
25
0,12
5
0,25 0,5 1
0,03
125
0,06
25
0,12
5
0,25 0,5 1
0,03
125
0,06
25
0,12
5
0,25 0,5 1
0,03
125
0,06
25
0,12
5
0,25 0,5 1
0,03
125
0,06
25
0,12
5
0,25 0,5 1
3125 ng/ml
625 ng/ml
125 ng/ml
25 ng/ml
5 ng/ml
1 ng/ml
Printed protein concentration in mg/ml
Slide H Slide P Slide E Slide AL Slide NC
0
200
400
600
800
1000
1200
1400
0,0
312
5
0,0
62
5
0,1
25
0,2
5
0,5 1
0,0
312
5
0,0
62
5
0,1
25
0,2
5
0,5 1
0,0
312
5
0,0
62
5
0,1
25
0,2
5
0,5 1
0,0
312
5
0,0
62
5
0,1
25
0,2
5
0,5 1
0,0
312
5
0,0
62
5
0,1
25
0,2
5
0,5 1
3125 ng/ml
625 ng/ml
125 ng/ml
25 ng/ml
5 ng/ml
1 ng/ml
colours refer to target con-centration (osteopontin)
S/B
rat
io
