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