General directions for the use of surfaces with amino and carboxylic groups

biomat has developed modified polystyrene surfaces introducing chemical groups such as NH/NH2 and COOH.

These groups are able to covalently bind compounds to the plastic surface. The optical properties of polystyrene remain unchanged, allowing to use the modified surfaces as powerful tools for diagnostic assays.

These surfaces offer the possibility to

Hereunder are some examples of application that can be used as guidelines to enable users to develop their own bio-specific assays.

1. Coupling of NHS-activated compounds

A very simple and easy application of biomat NH/NH2 surfaces is coupling of molecules that have been activated by esterification with N-hydroxysuccinimide derivatives (NHS). In our experiment a n-Hydroxysuccinimide active ester of biotin links immediately via its carbonilic group to the surface amino groups.

Fig. 1

Preparation of reagents and buffers

Materials

Solid phase: biomat plates MT02F-AM1 (primary amino groups)
MT02F-AM2 (secondary amino groups)
MT01F-HB (high binding capacity)
E-Caproylamido-biotin-N- hydroxysuccinimide ester (NHS- biotin) BIO-SPA Cat No. B002-61
Dimetilformamide (DMFO) Fluka Cat No. 40250
Tween® 20 Merck Cat No. 822184
Streptavidin BIO-SPA Cat. No. S002-60
Streptavidin-peroxidase conjugate BIO-SPA Cat. No. SB01-61
BSA Intergen Cat. No. 3100
TMB peroxidase substrate Kirkegard & Perry Cat. No. 50-76-05

NHS-Biotin stock solution
NHS-biotin 6mg
DMFO 2 ml

NHS-Biotin solution 150 µg/ml
NHS Biotin stock solution 500µl
PBS 0,1M pH 7,2-0,15% Tween® 20 ..to 10ml

NHS-Biotin solution 100µg/ml
NHS Biotin stock solution 333µl
PBS 0,1M pH 7,2-0,15% Tween® 20 ..to 10ml

NHS-Biotin solution 50µg/ml
NHS Biotin stock solution 167µl
PBS 0,1M pH 7,2-0,15% Tween® 20 ..to 10ml

NHS-Biotin solution 10µg/ml
NHS Biotin stock solution 33µl
PBS 0,1M pH 7,2-0,15% Tween® 20 ..to 10ml

Streptavidin-mix
Streptavidin 50µg
Streptavidin-peroxidase 1µg
PBS-BSA 1% 10ml

Experiment

  1. Add 100µl of NHS-biotin solutions 150-100-50-10 µg/ml and 0,1M PBS pH 7.2 + 0.15% Tween® 20 as 0 µg/ml to the wells (with primary amines and HB). Seal the wells with adhesive tape to prevent evaporation.
  2. Incubate overnight at room temperature
  3. Empty the wells and wash with 0.1M PBS pH 7.2 + 0.05% Tween® 20 four times
  4. Add 100µl of streptavidin mix to each well and incubate 30 minutes at room temperature
  5. Empty the wells and wash with 0,1M PBS pH 7.2 + 0.05% Tween® 20 four times
  6. Add 100 µl /well of TMB substrate solution and incubate for 10 minutes at room temperature
  7. Stop the substrate reaction by adding 100 µl of sulphuric acid 1 N and read the optical density values at 450 nm.

Results

The results show a clear correlation between the concentration of NHS-biotin added to the wells and the amount of biotin bound to the biomat NH2 surface.

On the other side no biotin is bound onto the plate without primary amino groups grafted to its surface, showing that passive adsorption of neither biotin nor enzyme conjugate occurs.

We therefore conclude that NHS-Biotin has indeed been covalently bound to the amino groups present on the biomat NH2 surface.

Fig. 2

2. Coupling hapten or peptide, having a carboxylic group, to biomat NH/NH2 surface

The carboxylic group presents in a molecule with a low molecular weight, such as a hapten or a peptide, binds to biomat NH/NH2 through formation of amide bonds between the carboxylic group presents in the molecule and the surface amino group by the combined action of carbodiimide and N-hydroxysuccinimide.

Reaction scheme for coupling of the hapten, biotin, through its available carboxylic group:

Fig. 3

Preparation of reagents and buffers

Materials

Solid phase: biomat plates MT02F-AM1 (primary amino groups)
MT01F-HB (high binding capacity)
d-Biotin Sigma Cat. No. B 4501
1-Ethyl-3-(3 dimethylaminopropyl)-carbodiimide (EDC) Sigma Cat. No. E 1769
Sulfo-N-hydroxysuccinimide (sulfo-NHS) Fluka Cat. No. 56485
Dimethylsulfoxide (DMSO) Merck Cat. No. 2931
Tween® 20 Merck Cat. No. 822184
Streptavidin BIO-SPA Cat. No. S002-60
Streptavidin-peroxidase conjugate BIO-SPA Cat. No. SB01-61
BSA Intergen Cat. No. 3100
TMB peroxidase substrate Kirkegard & Perry Cat. No. 50-76-05
NHS-Biotin stock solution
d-Biotin 7,8 mg
DMSO 0,5 ml
Distilled water 0,5 ml

Biotin/NHS solution
Biotin stock solution 500µl
Sulfo-NHS 3,45 mg.
Distilled water - 0,30% Tween® 20 to 10 ml

EDC solution
EDC 5,8 mg
Distilled water to 10 ml

Streptavidin-mix
Streptavidin-mix 50µl
Streptavidin-peroxidase 1µl
PBS-BSA 1% 10 ml

Experiment

  1. Add 50 µl of distilled water to each well, apart from wells in column 2. Then add 100 µl of Biotin-NHS solution to all wells in column 2.
  2. Dilute by transferring 50 µl from the wells in column 2 to column 3, mix, transfer 50 µl from column 3 to column 4, mix and proceed in this way up to column 12.
  3. To start reaction: add 50 µl of EDC solution to each column. In blank experiment (column 1) add 50 µl of distilled water instead of EDC.
  4. Incubate at room temperature for 2 hours.
  5. Empty the wells and wash with 0.1 M PBS pH 7.2 + 0.05% Tween® 20 four times.
  6. Add 100 µl of streptavidin mix to each well and incubate for 30 minutes at room temperature
  7. Empty the wells and wash with 0.1M PBS pH 7.2 + 0.05% Tween® 20 four times
  8. Add 100 µl of TMB substrate solution to each well and incubate for 10 minutes at room temperature.
  9. Stop the substrate reaction by adding 100 µl of sulphuric acid 1 N and read the optical density values at 450 nm.

Results

The results of this experiment clearly show that the molecule (biotin) is bound in a detectable way to the biomat NH2 (cod. AM1) whereas no detection could be obtained on biomat HB.

The results indicate that a covalent coupling has taken place between the carboxylic group in the biotin and the primary amino group grafted on the biomat NH2.

The results (data not displayed) point out that without adding carbodiimide the covalent binding of biotin does not occur.

Fig. 4

3. Coupling molecules, having an amino group, to biomat COOH surface

The amino group presents in any molecules, such as peptides or proteins, binds to biomat COOH through formation of amide bonds between the amino group presents in the molecule and the surface carboxylic group by the action of carbodiimide .

Reaction scheme for coupling of the hapten, biotin-hydrazide, through its available amino group:

Fig. 6

Preparation of reagents and buffers

Materials

Solid phase: biomat plates MT04F-COOH
MT01F-HB (high binding capacity)
Biotin-Hydrazide Sigma Cat. No. B 7639
1-Ethyl-3-(3 dimethylaminopropyl)-carbodiimide (EDC) Sigma Cat. No. E 1769
2-Morpholinoethanesulfonic acid (MES) Fluka Cat. No. 69889
Dimethylsulfoxide (DMSO) Merck Cat. No. 2931
Tween® 20 Merck Cat. No. 822184
Streptavidin BIO-SPA Cat. No. S002-60
Streptavidin-peroxidase conjugate BIO-SPA Cat. No. SB01-61
BSA Intergen Cat. No. 3100
TMB peroxidase substrate Kirkegard & Perry Cat. No. 50-76-05
Biotin-hydrazide stock solution
Biotin-hydrazide 5 mg
DMSO to 5 ml

Biotin-hydrazide solution 100 µg/ml
Biotin-hydrazide stock solution 1000 µl
EDC 10 mg
MES 0.1M pH 6,0 to 10 ml

Biotin-hydrazide solution 50 µg/ml
Biotin-hydrazide stock solution 500 µl
EDC 10 mg
MES 0.1M pH 6,0 10 mg

Biotin-hydrazide solution 10 µg/ml
Biotin-hydrazide stock solution 100 µl
EDC 10 mg
MES 0.1M pH 6,0 10 mg

Biotin-hydrazide solution 1,0 µg/ml
Biotin-hydrazide stock solution 10 µl
EDC 10 mg
MES 0.1M pH 6,0 10 mg

Biotin-hydrazide solution 0,5 µg/ml
Biotin-hydrazide stock solution 5 µl
EDC 10 mg
MES 0.1M pH 6,0 10 mg

Biotin-hydrazide solution 0,25 µg/ml
Biotin-hydrazide stock solution 2,5 µl
EDC 10 mg
MES 0.1M pH 6,0 10 mg

Biotin-hydrazide solution 0,1 µg/ml
Biotin-hydrazide stock solution 1,0 µl
EDC 10 mg
MES 0.1M pH 6,0 10 mg

Streptavidin-mix
Streptavidin 50 µg
Streptavidin-peroxidase 0,5 µg
PBS-BSA 1% 10 ml

Experiment

  1. Add 100 µl of biotin-hydrazide solutions 100-50-10-1-0,5-0,25-0,1 µg/ml and 100 µl 0,1 M MES pH 6,0 as 0 µg/ml to the wells (carboxylated and HB not activated). Seal the wells with adhesive tape to prevent evaporation.
  2. Incubate overnight at room temperature.
  3. Empty the wells and wash with 0.1 M PBS pH 7.2 + 0.05% Tween® 20 four times.
  4. Add 100 µl of streptavidin- mix to each well and incubate for 30 minutes at room temperature.
  5. Empty the wells and wash with 0.1 M PBSpH 7.2 + 0.05% Tween® 20 four times.
  6. Add 100 µl of TMB substrate solution to each well and incubate for 10 minutes at room temperature.
  7. Stop the substrate reaction by adding 100 µl of sulphuric acid 1 N and read the optical density values at 450 nm.

Results

The results of this experiment clearly show that the molecule (biotin-hydrazide) is bound in a detectable way to biomat COOH whereas no detection could be obtained on biomat HB.

The results indicate that a covalent coupling has taken place between the amino group in the biotin-hydrazide and the carboxylic group grafted on the biomat COOH.

The results (data not displayed) show that without adding carbodiimide the covalent binding of biotin-hydrazide does not occur.

Fig. 5

Aminated Surfaces